Methods of treating cancer using immunostimulatory oligonucleotides

ABSTRACT

Nucleic acid sequences containing unmethylated CpG dinucleotides that modulate an immune response including stimulating a Th1 pattern of immune activation, cytokine production, NK lytic activity, and B cell proliferation are disclosed. The sequences are also useful as a synthetic adjuvant.

RELATED APPLICATION

[0001] This application is a continuation of co-pending U.S. Ser. No.09/337,619, filed Jun. 21, 1999, which is a divisional of U.S. Ser. No.08/960,774, filed Oct. 30, 1997, now issued as U.S. Pat. No. 6,239,116B1on May 29, 2001, which is a continuation-in-part of U.S. Ser. No.08/738,652, filed Oct. 30, 1996, now issued as U.S. Pat. No. 6,207,646B1on Mar. 27, 2001, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/386,063, filed Feb. 7, 1995, now issued as U.S.Pat. No. 6,194,388B1 on Feb. 27, 2001, which is a continuation-in-partof U.S. patent application Ser. No. 08/276,358, filed Jul. 15, 1994which is now abandoned, each of which are incorporated herein byreference in their entirety.

GOVERNMENT

[0002] The work resulting in this invention was supported in part byNational Institute of Health Grant No. R29-AR42556-01. The U.S.Government may have rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to oligonucleotides andmore specifically to oligonucleotides which have a sequence including atleast one unmethylated CpG dinucleotide which are immunostimulatory.

BACKGROUND OF THE INVENTION

[0004] In the 1970s, several investigators reported the binding of highmolecular weight DNA to cell membranes (Lerner, R. A., et al. 1971.“Membrane-associated DNA in the cytoplasm of diploid human lymphocytes.”Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K., R. W. Wagner, P. K.McAllister, and B. Rosenberg. 1975. “Cell-surface-associated nucleicacid in tumorigenic cells made visible with platinum-pyrimidinecomplexes by electron microscopy.” Proc. Natl. Acad. Sci. USA 72:928).In 1985, Bennett et al. presented the first evidence that DNA binding tolymphocytes is similar to a ligand receptor interaction: binding issaturable, competitive, and leads to DNA endocytosis and degradationinto oligonucleotides (Bennett, R. M., G. T. Gabor, and M. M. Merritt,1985. “J. Clin. Invest. 76:2182). Like DNA, oligodeoxyribonucleotides(ODNs) are able to enter cells in a saturable, sequence independent, andtemperature and energy dependent fashion (reviewed in Jaroszewski, J.W., and J. S. Cohen. 1991. “Cellular uptake of antisenseoligodeoxynucleotides.” Advanced Drug Deliver Reviews 6:235; Akhtar, S.,Y. Shoji, and R. L. Juliano. 1992. “Pharmaceutical aspects of thebiological stability and membrane transport characteristics of antisenseoligonucleotides.” In: Gene Regulation: Biology of Antisense RNA andDNA. R. P. Erickson, and J. G. Izant, eds. Raven Press, Ltd. New York,pp. 133; and Zhao, Q., T. Waldschmidt, E. Fisher, C. J. Herrera, and A.M. Krieg. 1994. “Stage specific oligonucleotide uptake in murine bonemarrow B cell precursors.” Blood 84:3660). No receptor for DNA or ODNuptake has yet been cloned, and it is not yet clear whether ODN bindingand cell uptake occurs through the same or a different mechanism fromthat of high molecular weight DNA.

[0005] Lymphocyte ODN uptake has been shown to be regulated by cellactivation. Spleen cells stimulated with the B cell mitogen LPS haddramatically enhanced ODN uptake in the B cell population, while spleencells treated with the T cell mitogen Con A showed enhanced ODN uptakeby T but not B cells (Krieg, A. M., F. Ginelig-Meyling, M. F. Gourley,W. J. Kisch, L. A. Chrisey, and A. D. Steinberg. 1991. “Uptake ofoligodeoxyribonucleotides by lymphoid cells is heterogeneous andinducible.” Antisense Research and Development 1:161).

[0006] Several polynucleotides have been extensively evaluated asbiological response modifiers. Perhaps the best example is poly (I,C)which is a potent inducer of IFN production as well as macrophageactivator and inducer of NK activity (Talmadge, J. E., J. Adams, H.Phillips, M. Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R.H. Wiltrout, and M. A. Chirigos. 1985. “Immunomodulatory effects in miceof polyinosinic-polycytidylic acid complexed with poly-L-lysine andcarboxymethylcellulose.” Cancer Res. 45:1058; Wiltrout, R. H., R. R.Salup, T. A. Twilley, and J. E. Talnadge. 1985. “Immunomodulation ofnatural killer activity by polyribonucleotides.” J. Biol. Respn. Mod.4:512; Krown, S. E. 1986. “Interferons and interferon inducers in cancertreatment.” Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp,J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W.G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P.Creekmore. 1992. “Polyinosinic-polycytidylic acid complexed withpoly-L-lysine and carboxymethylcellulose in combination withinterleukin-2 in patients with cancer: clinical and immunologicaleffects.” Canc. Res. 52:3005). It appears that this murine NK activationmay be due solely to induction of IFN-β secretion (Ishikawa, R., and C.A. Biron. 1993. “IFN inducation and associated changes in splenicleukocyte distribution”. J. Immunol. 150:3713). This activation wasspecific for the robose sugar since deoxyribose was ineffective. Itspotent in vitro antitumor activity led to several clinical trials usingpoly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (toreduce degradation by RNAse) Talmadge, J. E., et al., 1985. cited supra;Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986. citedsupra); and Ewel, C. H., et al., 1992. cited supra). Unfortunately,toxic side effects have thus far prevented poly (I,C) from becoming auseful therapeutic agent.

[0007] Guanine ribonucleotides substituted at the C8 position witheither a bromine or a thiol group are B cell mitogens and may replace “Bcell differentiation factors” (Feldbush, T. L., and Z. K., Ballas. 1985.“Lymphokine-like activity of 8-mercaptoguanosine: induction of T and Bcell differentiation.” J. Immunol. 134:3204; and Goodman, M. G. 1986.“Mechanism of synergy between T cell signals and C8-substituted guaninenucleosides in humoral immunity: B lymphotropic cytokines induceresponsiveness to 8-mercaptoguanosine.” J. Immunol. 136:3335).8-mercaptoguanosine and 8-bromoguanosine also can substitute for thecytokine requirement for the generation of MHC restricted CTL (Feldbush,T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E.Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activationof murine natural killer cells and macrophages by 8bromoguanosine.” J.Immunol. 140:3249), and synergize with IL-2 in inducing murine LAKgeneration (Thompson, R. A., and Z. K. Ballas. 1990.“Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as anIL-2-sparing agent in LAK generation.” J. Immunol. 145:3524). The NK andLAK augmenting activities of these C8-substituted guanosines appear tobe due to their induction of IFN (Thompson, R. A., et al. 1990. citedsupra0. Recently, a 5′ triphosphorylated thymidine produced by amycobacterium was found to be mitogenic for a subset of human γδ T cells(Constant, P., F. Davodeau, M.-A. Peyrat, Y. Poquet, G. Puzo, M.Bonneville, and J.-J. Fournie. 1994. “Stimulation of human γδ T cells bynonpeptidic mycobacterial ligands.” Science 264:267). This reportindicated the possibility that the immune system may have evolved waysto preferentially respond to microbial nucleic acids.

[0008] Several observations suggest that certain DNA structures may alsohave the potential to activate lymphocytes. For example, Bell et al.reported that nucleosoinal protein-DNA complexes (but not naked DNA) inspleen cell supernatants caused B cell proliferation and immunoglobulinsecretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990.“Immunogenic DNA-related factors.” J. Clin. Invest. 85:1487). In othercases, naked DNA has been reported to have immune effects. For example,Messina et al. have recently reported that 260 to 800 bp fragments ofpoly (dG)•(dC) and poly (dG•dC) were mitogenic for B cells (Messina, J.P., G. S. Gilkeson, and D. S. Piesetsky. 1993. “The influence of DNAstructure on the in vitro stimulation of murine lymphocytes by naturaland synthetic polynucleotide antigens.” Cell. Immunol. 147:148).Tokunaga, et al. have reported that dG•dC induces γ-IFN and NK activity(Tokunaga, S. Yamamoto, and K. Nama. 1988. “A synthetic single-strandedDNA, poly(dG, dC), induces interferon-α/β and -γ, augments naturalkiller activity, and suppresses tumor growth.” Jpn. J. Cancer Res.79:682). Aside from such artificial homopolymer sequences, Pisetsky etal. reported that pure mammalian DNA has no detectable immune effects,but that DNA from certain bacteria induces B cell activation andimmunoglobulin secretion (Messina, J. P., G. S. Gilkeson, and D. S.Pisetsky. 1991. “Stimulation of in vitro murine lymphocyte proliferationby bacterial DNA.” J. Immunol. 147:1759). Assuming that these data didnot result from some unusual contaminant, these studies suggested that aparticular structure or other characteristic of bacterial DNA renders itcapable of triggering B cell activation. Investigations of mycobacterialDNA sequences have demonstrated that ODN which contain certainpalindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto,T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. “Uniquepalindromic sequences in synthetic oligonucleotides are required toinduce INF and augment INF-mediated natural killer activity.” J.Immunol. 148:4072; Kuramoto, E., 0. Yano, Y. Kimura, M. Baba, T. Makino,S. Yamamoto, T. Yamamoto, T. Kataoka, and T. Tokunaga. 1992.“Oligonucleotide sequences required for natural killer cell activation.”Jpn. J. Cancer Res. 83:1128).

[0009] Several phosphorothioate modified ODN have been reported toinduce in vitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu,and W. E. Paul. 1992. “An antisense oligonucleotide complementary to asequence in Iγ2b increases γ2b germline transcripts, stimulates B cellDNA synthesis, and inhibits immunoglobulin secretion.” J. Exp. Med.175:597; Mcintyre, K. W., K. Lombard-Gillooly, J. R. Perez, C. Kunsch,U. M. Sarmiento, J. D. Larigan, K. T. Landreth, and R. Narayanan. 1993.“A sense phosphorothioate oligonucleotide directed to the initiationcodon of transcription factor NF-κB T65 causes sequence-specific immunestimulation.” Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C.F. Reich. 1993. “Stimulation of murine lymphocyte proliferation by aphosphorothioate oligonucleotide with antisense activity for herpessimplex virus.” Life Sciences 54:101). These reports do not suggest acommon structural motif or sequence element in these ODN that mightexplain their effects.

[0010] The cAMP response element binding protein (CREB) and activatingtranscription factor (ATF) or CREB/ATF family of transcription factorsis a ubiquitously expressed class of transcription factors of which 11members have so far been cloned (reviewed on de Groot, R. P., and P.Sassone-Corsi: “Hormonal control of gene expression: Multiplicity andversatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclearregulators.” Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson:“Transcriptional regulation by CREB and its relatives.” Biochim.Biophys. Acta 1174:221, 1993). They all belong to the basicregion/leucine zipper (bZip) class of proteins. All cells appear toexpress one or more CREB/ATF proteins, but the members expressed and theregulation of mRNA splicing appear to be tissue-specific. Differentialsplicing of activation domains can determine whether a particularCREB/ATF protein will be a transcriptional inhibitor or activator. ManyCREB/ATF proteins activate viral transcription, but some splicingvariants which lack the activation domain are inhibitory. CREB/ATFproteins can bind DNA as homo- or hetero-dimers through the cAMPresponse element, the CRE, the consensus form of which is theunmethylated sequence TGACGTC (SEQ. ID. No. 103) (binding is abolishedif the CpG is methylated) (Iguchi-Ariga, S. M. M., and W. Schaffner:“CpG methylation of the cAMP-responsive enhancer/promoter sequenceTGACGTCA (SEQ. ID. No. 104) abolishes specific factor binding as well astranscriptional activation.” Genese & Develop. 3:612, 1989.

[0011] The transcriptional activity of the CRE is increased during Bcell activation (Xie. H., T. C. Chiles, and T. L. Rothstein: “Inductionof CREB activity via the surface Ig receptor of B cells.” J. Immunol.151:880, 1993). CREB/ATF proteins appear to regulate the expression ofmultiple genes through the CRE including immunologically important genessuch as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R.Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 andCREB recognize a common essential site in the human prointerleukin 1βgene.” Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D.Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: “Antisense DNAinhibition of tumor growth induced by c-Ha-ras oncogene in nude mice.”Cancer Res. 53:577, 1993), IFN- (Du, W., and T. Maniatis: “An ATF/CREBbinding site protein is required for virus induction of the humaninterferon β gene.” Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-1(Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: “Binding ofAP-1/CREB proteins and of MDBP to contiguous sites downstream of thehuman TGF-β1 gene.” Biochim. Biophys. Acta 1219:55, 1994), TGF-2, classII MHC (Cox, P. M., and C. R. Goding: “An ATF/CREB binding motif isrequired for aberrant constitutive expression of the MHC class II DRαpromoter and activation by SV40 T-antigen.” Nucl. Acids Res. 20:4881,1992), E-selectin, GM-CSF, CD-8, the germline Ig constant region gene,the TCR V gene, and the proliferating cell nuclear antigen (Huang, D.,P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M. B.Prystowsky: “Promoter activity of the proliferating-cell nuclear antigengene is associated with inducible CRE-binding proteins in interleukin2-stimulated T lymphocytes.” Mol. Cell. Biol. 14:4233, 1994). Inaddition to activation through the cAMP pathway, CREB can also mediatetranscriptional responses to changes in intracellular Ca⁺⁺ concentration(Sheng, M., G. McFadden, and M. E. Greenberg: “Membrane depolarizationand calcium induce c-fos transcription via phosphorylation oftranscription factor CREB.” Neuron 4:571, 1990).

[0012] The role of protein-protein interactions in transcriptionalactivation by CREB/ATF proteins appears to be extremely important. Thereare several published studies reporting direct or indirect interactionsbetween NFKB proteins and CREB/ATF proteins (Whitley, et al., (1994)Mol. & Cell. Biol. 14:6464; Cogswell, et al., (1994) J. Immun. 153:712;Hines, et al., (1993) Oncogene 8:3189; and Du, et al., (1993) Cell74:887. Activation of CREB through the cyclic AMP pathway requiresprotein kinase A (PKA), which phosphorylates CREB³⁴¹ on ser¹³³ andallows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J.R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G.Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman: “Nuclearprotein CBP is a coactivator for the transcription factor CREB.” Nature370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T.Sinea, M. Karin, J. Feramisco, and M. Montminy: “Activation of cAMP andmitogen responsive genes relies on a common nuclear factor.” Nature370:226, 1994). CBP in turn interacts with the basal transcriptionfactor TFIIB causing increased transcription. CREB also has beenreported to interact with dTAFII 110, a TATA binding protein-associatedfactor whose binding may regulate transcription (Ferreri, K., G. Gill,and M. Montminy: “The cAMP-regulated transcription factor CREB interactswith a component of the TFIID complex.” Proc. Natl. Acad. Sci. USA91:1210, 1994). In addition to these interactions, CREB/ATF proteins canspecifically bind multiple other nuclear factors (Hoeffler, J. P., J. W.Lustbadfer, and C.-Y. Chen: “Identification of multiple nuclear factorsthat interact with cyclic adenosine 3′,5′-monophosphate responseelement-binding protein and activating transcription factor-2 byprotein-protein interactions.” Mol. Endocrinol. 5:256, 1991) but thebiologic significance of most of these interactions is unknown. CREB isnormally thought to bind DNA either as a homodimer or as a heterodimerwith several other proteins. Surprisingly, CREB monomers constitutivelyactivate transcription (Krajewski, W., and K. A. W. Lee: “A monomericderivative of the cellular transcription factor CREB functions as aconstitutive activator.” Mol. Cell. Biol. 14:7204, 1994).

[0013] Aside from their critical role in regulating cellulartranscription, it has recently been shown that CREB/ATF proteins aresubverted by some infectious viruses and retroviruses, which requirethem for viral replication. For example, the cytomegalovirus immediateearly promoter, one of the strongest known mammalian promoters, containseleven copies of the CRE which are essential for promoter function(Chang, Y.-N., S. Crawford, J. Stall, D. R. Rawlins, K.-T. Jeang, and G.S. Hayward: “The palindromic series 1 repeats in the simiancytomegalovirus major immediate-early promoter behave as both strongbasal enhancers and cyclic AMP response elements.” J. Virol. 64:264,1990). At least some of the transcriptional activating effects of theadenovirus E1A protein, which induces many promoters, are due to itsbinding to the DNA binding domain of the CREB/ATF protein, ATF-2, whichmediates E1A inducible transcription activation (Liu, F., and M. R.Green: “Promoter targeting by adenovirus E1A through interaction withdifferent cellular DNA-binding domains.” Nature 368:520, 1994). It hasalso been suggested that E1A binds to the CREB-binding protein, CBP(Arany, Z., W. R. Sellers, D. M. Livingston, and R. Eckner:“E1A-associated p300 and CREB-associated CBP belong to a conservedfamily of coactivators.” Cell 77:799, 1994). Human T lymphotropicvirus-I (HTLV-1), the retrovirus which causes human T cell leukemia andtropical spastic paresis, also requires CREB/ATF proteins forreplication. In this case, the retrovirus produces a protein, Tax, whichbinds to CREB/ATF proteins and redirects them from their normal cellularbinding sites to different DNA sequences (flanked by G- and G-richsequences) present within the HTLV transcriptional enhancer(Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J. V. Cross, B. R. Cullen,I. M. Boros, and C.-Z. Giam: “In vitro selection of DNA elements highlyresponsive to the human T-cell lymphotropic virus type 1 transcriptionalactivator, Tax.” Mol. Cell. Biol. 14:456, 1994; Adya, N., L.-J. Zhao, W.Huang, I. Boros, and C.-Z. Giam: “Expansion of CREB's DNA recognitionspecificity by Tax results from interaction with Ala-Ala-Arg atpositions 282-284 near the conserved DNA-binding domain of CREB.” Proc.Natl. Acad. Sci. USA 91:5642, 1994).

SUMMARY OF THE INVENTION

[0014] The present invention is based on the finding that certainnucleic acids containing unmethylated cytosine-guanine (CpG)dinucleotides activate lymphocytes in a subject and redirect a subject'simmune response from a Th2 to a Th1 (e.g., by inducing monocytic cellsand other cells to produce Th1 cytokines, including IL-12, IFN-γ andGM-CSF). Based on this finding, the invention features, in one aspect,novel immunostimulatory nucleic acid compositions.

[0015] In one embodiment, the invention provides an isolatedimmunostimulatory nucleic acid sequence containing a CpG motifrepresented by the formula:

5′N ₁ X ₁ CGX ₂ N ₂ 3′

[0016] wherein at least one nucleotide separates consecutive CpGs; X₁ isadenine, guanine, or thymine; X₂ is cytosine or thymine; N is anynucleotide and N₁+N₂ is from about 0-26 bases with the proviso that N₁and N₂ do not contain a CCGG quadmer or more than one CCG or CGG trimer;and the nucleic acid sequence is from about 8-30 bases in length.

[0017] In another embodiment, the invention provides an isolatedimmunostimulatory nucleic acid sequence contains a CpG motif representedby the formula:

5′ N ₁ X ₁ X ₂ CGX ₃ X ₄ N ₂ 3′

[0018] wherein at least one nucleotide separates consecutive CpGs; X₁X₂is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X₃X₄ is selected from the group consisting of TpT or CpT; N is anynucleotide and N₁+N₂ is from about 0-26 bases with the proviso that N₁and N₂ do not contain a CCGG quadmer or more than one CCG or CGG trimer;and the nucleic acid sequence is from about 8-30 bases in length.

[0019] In another embodiment, the invention provides a method ofstimulating immune activation by administering the nucleic acidsequences of the invention to a subject, preferably a human. In apreferred embodiment, the immune activation effects predominantly a Th1pattern of immune activation.

[0020] In another embodiment, the nucleic acid sequences of theinvention stimulate cytokine production. In particular, cytokines suchas IL-6, IL-12, IFN-γ, TNF-α and GM-CSF are produced via stimulation ofthe immune system using the nucleic acid sequences described herein. Inanother aspect, the nucleic acid sequences of the invention stimulatethe lytic activity of natural killer cells (NK) and the proliferation ofB cells.

[0021] In another embodiment, the nucleic acid sequences of theinvention are useful as an artificial adjuvant for use during antibodygeneration in a mammal such as a mouse or a human.

[0022] In another embodiment, autoimmune disorders are treated byinhibiting a subject's response to CpG mediated leukocyte activation.The invention provides administration of inhibitors of endosomalacidification such as bafilomycin a, chloroquine, and monensin toameliorate autoimmune disorders. In particular, systemic lupuserythematosus is treated in this manner.

[0023] The nucleic acid sequences of the invention can also be used totreat, prevent or ameliorate other disorders (e.g., a tumor or cancer ora viral, fungal, bacterial or parasitic infection). In addition, thenucleic acid sequences can be administered to stimulate a subject'sresponse to a vaccine. Furthermore, by redirecting a subject's immuneresponse from Th2 to Th1, the claimed nucleic acid sequences can be usedto treat or prevent an asthmatic disorder. In addition, the claimednucleic acid molecules can be administered to a subject in conjunctionwith a particular allergen as a type of desensitization therapy to treator prevent the occurrence of an allergic reaction associated with anasthmatic disorder.

[0024] Further, the ability of the nucleic acid sequences of theinvention described herein to induce leukemic cells to enter the cellcycle supports their use in treating leukemia by increasing thesensitivity of chronic leukemia cells followed by conventional ablativechemotherapy, or by combining the nucleic acid sequences with otherimmunotherapies.

[0025] Other features and advantages of the invention will become moreapparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0026] FIGS. 1A-C are graphs plotting dose-dependent IL-6 production inresponse to various DNA sequences in T cell depleted spleen cellcultures.

[0027] FIG. A1. E. coli DNA () and calf thymus DNA (▪) sequences andLPS (at 10× the concentration of E. coli and calf thymus DNA) (♦).

[0028]FIG. 1B. Control phosphodiester oligodeoxynucleotide (ODN) 5′ATGGAAGGTCCAGTGTTCTC 3′ (SEQ ID NO: 114) (▪) and two phosphodiester CpGODN 5′ ATCGACCTACGTGCGTTCTC 3′ (SEQ ID NO: 2) (♦) and 5′TCCATAACGTTCCTGATGCT 3′ (SEQ ID NO: 3) ().

[0029]FIG. 1C. Control phosphorothioate ODN 5′ GCTAGATGTTAGCGT 3′ (SEQID NO: 4) (▪) and two phosphorothioate CpG ODN 5′ GAGAACGTCGACCTTCGAT 3′(SEQ ID NO: 5) (♦) and 5′ GCATGACGTTGAGCT 3′ (SEQ ID NO: 6) (). Datapresent the mean±standard deviation of triplicates.

[0030]FIG. 2 is a graph plotting IL-6 production induced by CpG DNA invivo as determined 1-8 hrs after injection. Data represent the mean fromduplicate analyses of sera from two mice. BALB/c mice (two mice/group)were injected iv. with 100 μl of PBS (□) of 200 μg of CpGphosphorothioate ODN 5′TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO: 7) (▪) ornon-CpG phosphorothioate ODN 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO: 8(♦).

[0031]FIG. 3 is an autoradiograph showing IL-6 mRNA expression asdetermined by reverse transcription polymerase chain reaction in liver,spleen, and thymus at various time periods after in vivo stimulation ofBALB/c mice (two mice/group) injected iv with 100 μl of PBS, 200 μg ofCpG phosphorothioate ODN 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO: 7) ornon-CpG phosphorothioate ODN 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO: 8).

[0032]FIG. 4A is a graph plotting dose-dependent inhibition ofCpG-induced IgM production by anti-IL-6. Splenic B-cells from DBA/2 micewere stimulated with CpG ODN 5′ TCCAAGACGTTCCTGATGCT 3′ (SEQ ID NO: 9)in the presence of the indicated concentrations of neutralizinganti-IL-6 (♦) or isotype control Ab () and IgM levels in culturesupernatants determined by ELISA. In the absence of CpG ODN, theanti-IL-6 Ab had no effect on IgM secretion (▪).

[0033]FIG. 4B is a graph plotting the stimulation index of CpG-inducedsplenic B cells cultured with anti-L-6 and CpG S-ODN 5′TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO: 7)(♦) or anti-IL-6 antibody only(▪). Data present the mean ± standard deviation of triplicates.

[0034]FIG. 5 is a bar graph plotting chloramphenicol acetyltransferase(CAT) activity in WEHI-231 cells transfected with a promoter-less CATconstruct (pCAT), positive control plasmid (RSV), or IL-6 promoter-CATconstruct alone or cultured with CpG 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ IDNO: 7) or non-CpG 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO: 8)phosphorothioate ODN at the indicated concentrations. Data present themean of triplicates.

[0035]FIG. 6 is a schematic overview of the immune effects of theimmunostimulatory unmethylated CpG containing nucleic acids, which candirectly activate both B cells and monocytic cells (includingmacrophages and dendritic cells) as shown. The immunostimulatoryoligonucleotides do not directly activate purified NK cells, but renderthem competent to respond to IL-12 with a marked increase in their IFN-γsecretion by NK cells, the immunostimulatory nucleic acids promote a Th1type immune response. No direct activation of proliferation of cytokinesecretion by highly purified T cells has been found. However, theinduction of Th1 cytokine secretion by the immunostimulatoryoligonucleotides promotes the development of a cytotoxic lymphocyteresponse.

[0036]FIG. 7 is an autoradiograph showing NFκB mRNA induction inmonocytes treated with E. coli (EC) DNA (containing unmethylated CpGmotifs), control (CT) DNA (containing no unmethylated CpG motifs) andlipopolysaccharide (LPS) at various measured times, 15 and 30 minutesafter contact.

[0037]FIG. 8A shows the results from a flow cytometry study using mouseB cells with the dihydrorhodamine 123 dye to determine levels ofreactive oxygen species. The dye only sample in Panel A of the figureshows the background level of cells positive for the dye at 28.6%. Thislevel of reactive oxygen species was greatly increased to 80% in thecells treated for 20 minutes with PMA and ionomycin, a positive control(Panel B). The cells treated with the CpG oligo (TCCATGACGTTCCTGACGTTSEQ ID NO: 10) also showed an increase in the level of reactive oxygenspecies such that more than 50% of the cells became positive (Panel D).However, cells treated with an oligonucleotide that lacked a CpG motif(TCCATGAGCTTCCTGAGTCT SEQ ID NO: 8) did not show this significantincrease in the level of reactive oxygen species (Panel E).

[0038]FIG. 8B shows the results from a flow cytometry study using mouseB cells in the presence of chloroquine with the dihydrorhodamine 123 dyeto determine levels of reactive oxygen species. Chloroquine slightlylowers the background level of reactive oxygen species in the cells suchthat the untreated cells in Panel A have only 4.3% that are positive.Chloroquine completely abolishes the induction of reactive oxygenspecies in the cells treated with CpG DNA (Panel B) but does not reducethe level of reactive oxygen species in the cells treated with PMA andionomycin (Panel E).

[0039]FIG. 9 is a graph plotting lung lavage cell count over time. Thegraph shows that when the mice are initially injected with Schistosomamansoni eggs “egg”, which induces a Th2 immune response, andsubsequently inhale Schistosoma mansoni egg antigen “SEA” (open circle),many inflammatory cells are present in the lungs. However, when the miceare initially given CpG oligo (SEQ ID NO: 10) along with egg, theinflammatory cells in the lung are not increased by subsequentinhalation of SEA (open triangles).

[0040]FIG. 10 is a graph plotting lung lavage eosinophil count overtime. Again, the graph shows that when the mice are initially injectedwith egg and subsequently inhale SEA (open circle), many eosinophils arepresent in the lungs. However, when the mice are initially given CpGoligo (SEQ ID NO: 10) along with egg, the inflammatory cells in the lungare not increased by subsequent inhalation of the SEA (open triangles).

[0041]FIG. 11 is a bar graph plotting the effect on the percentage ofmacrophage, lymphocyte, neutrophil and eosinophil cells induced byexposure to saline alone; egg, then SEA; egg and SEQ ID NO: 10, thenSEA; and egg and control oligo (SEQ ID NO: 8), then SEA. When the miceare treated with the control oligo at the time of the initial exposureto the egg, there is little effect on the subsequent influx ofeosinophils into the lungs after inhalation of SEA. Thus, when miceinhale the eggs on days 14 or 21, they develop an acute inflammatoryresponse in the lungs. However, giving a CpG oligo along with the eggsat the time of initial antigen exposure on days 0 and 7 almostcompletely abolishes the increase in eosinophils when the mice inhalethe egg antigen on day 14.

[0042]FIG. 12 is a bar graph plotting eosinophil count in response toinjection of various amounts of the protective oligo SEQ ID NO: 10.

[0043]FIG. 13 is a graph plotting interleukin 4 (IL-4) production(pg/ml) in mice over time in response to injection of egg, then SEA(open diamond); egg and SEQ ID NO: 10, then SEA (open circle); orsaline, then saline (open square). The graph shows that the resultantinflammatory response correlates with the levels of the Th2 cytokineIL-4 in the lung.

[0044]FIG. 14 is a bar graph plotting interleukin 12 (IL-12) production(pg/ml) in mice over time in response to injection of saline; egg, thenSEA; or SEQ ID NO. 10 and egg, then SEA. The graph shows thatadministration of an oligonucleotide containing an unmethylated CpGmotif can actually redirect the cytokine response of the lung toproduction of IL-12, indicating a Th1 type of immune response.

[0045]FIG. 15 is a bar graph plotting interferon gamma (IFN-γ)production (pg/ml) in mice over time in response to injection of saline;egg, then saline; or SEQ ID NO: 10 and egg, then SEA. The graph showsthat administration of an oligonucleotide containing an unmethylated CpGmotif can also redirect the cytokine response of the lung to productionof IFN-γ, indicating a Th1 type of immune response.

DETAILED DESCRIPTION OF THE INVENTION

[0046] Definitions

[0047] As used herein, the following terms and phrases shall have themeanings set forth below:

[0048] An “allergen” refers to a substance that can induce an allergicasthmatic response in a susceptible subject. The list of allergens isenormous and can include pollens, insect venoms, animal dander dust,fungal spores and drugs (e.g., penicillin). Examples of natural, animaland plant allergens include proteins specific to the following genuses:Canine (Canis familiaris); Dermatophagoides (e.g., Dermatophagoidesfarinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia;Lolium (e.g., Lolium perenne or Lolium multifloruni); Cryptomeria(Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus(Alnus gultinosa); Betula (Betula verrucosa); Quercus (quercus alba);Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g.,Plantago lanceolata); Parietaria (e.g., Parietaria officinalis orParietaria judaica); Blattella (e.g., Blattella germanica), Apis (e.g.,Apis multiflorum); Cupressus (e.g., Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g., Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei); Thuya (e.g., Thuya orientalis), Chamaecyparis (e.g.,Chamaecyparis obtusa); Periplaneta (e.g., Periplaneta americana);Agropyron (e.g., Agropyron repens); Secale (e.g., Secale cereale);Triticum (e.g., Triticum aestivum); Dactylis (e.g., Dactylis glomerata);Festuca (e.g., Festuca elatior); Poa (e.g., Poa pratensis or Poacompressa); Avena (e.g., Avena saliva); Holcus (e.g., Holcus lanatus);Anthoxanthum (e.g., Anthoxanthum odoratum); Arrhenatherum (e.g.,Arrhenatherum elatius); Agrostis (e.g., Agrostis alba); Phleiun (e.g.,Phleum pratense); Phalaris (e.g., Phalaris arundinacea), Paspalum (e.g.,Paspalum notatum); Sorghum (e.g., Sorghum halepensis) and Bronils (e.g.,Bromus inermis).

[0049] An “allergy” refers to acquired hypersensitivity to a substance(allergen). Allergic conditions include eczema, allergic rhinitis orcoryza, hay fever, bronchial asthma, urticaria (hives) and foodallergies, and other atopic conditions.

[0050] “Asthma” refers to a disorder of the respiratory systemcharacterized by inflammation, narrowing of the airways and increasedreactivity of the airways to inhaled agents. Asthma is frequently,although not exclusively associated with atopic or allergic symptoms.

[0051] An “immune system deficiency” shall mean a disease or disorder inwhich the subject's immune system is not functioning in normal capacityor in which it would be useful to boost a subject's immune response forexample to eliminate a tumor or cancer (e.g., tumors of the brain, lung(e.g., small cell and non-small cells), ovary, breast, prostate, colon,as well as other carcinomas and sarcomas) or an infection in a subject.

[0052] Examples of infectious virus include: Retroviridae (e.g., humanimmunodeficiency viruses, such as HIV-1, also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fevervirus); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herperviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses); Poxyiridae (variola virsues, vaccinia viruses, pox viruses);and Iridoviridae (e.g., African swine fever virus); and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatitides (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1—internally transmitted; class 2—parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

[0053] Examples of infectious bacteria include: Helicobacter pyloris,Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M.tuberculosis, M. avium, M. Intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter erogenes, Klebsiella pneuomiae, Pasturellamulticoda, Bacteroides sp., Fusobacterium nucleatum, Sreptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomeyces israelli.

[0054] Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium falciparumand Toxoplasma gondii.

[0055] An “immunostimulatory nucleic acid molecule” refers to a nucleicacid molecule, which contains an unmethylated cytosine, guaninedinucleotide sequence (i.e., “CpG DNA” or DNA containing a cytosinefollowed by guanosine and linked by a phosphate bond) and stimulates(e.g., has a motogenic effect on, or induces or increases cytokineexpression by) a vertebrate lymphocyte. An immunostimulatory nucleicacid molecule can be double-stranded or single-stranded. Generally,double-stranded molecules are more stable in vivo, while single-strandedmolecules have increased immune activity.

[0056] In one preferred embodiment, the invention provides an isolatedimmunostimulatory nucleic acid sequence containing a CpG motifrepresented by the formula:

5′N ₁ X ₁ CGX ₂N₂3′

[0057] wherein at least one nucleotide separates consecutive CpGs; X₁ isadenine, guanine, or thymine; X₂ is cytosine or thymine; N is anynucleotide and N₁+N₂ is from about 0-26 bases with the proviso that N₁and N₂ do not contain a CCGG quadmer or more than one CCG or CGG trimer;and the nucleic acid sequence is from about 8-30 bases in length.

[0058] In another embodiment the invention provides an isolatedimmunostimulatory nucleic acid sequence contains a CpG motif representedby the formula:

5′N ₁ X ₁ X ₂ CGX ₃ X ₄ N ₂3′

[0059] wherein at least one nucleotide separates consecutive CpGs; X₁X₂is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X₃X₄ is selected from the group consisting of TpT or CpT; N is anynucleotide and N₁+N₂ is from about 0-26 bases with the proviso that N₁and N₂ do not contain a CCGG quadmer or more than one CCG or CGG trimer;and the nucleic acid sequence is from about 8-30 bases in length.

[0060] Preferably, the immunostimulatory nucleic acid sequences of theinvention include X₁X₂ selected from the group consisting of GpT, GpG,GpA and ApA and X₃X₄ is selected from the group consisting of TpT, CpTand GpT (see for example, Table 5). For facilitating uptake into cells,CpG containing immunostimulatory nucleic acid molecules are preferablyin the range of 8 to 30 bases in length. However, nucleic acids of anysize (even many kb long) are immunostimulatory if sufficientimmunostimulatory motifs are present, since such larger nucleic acidsare degraded into oligonucleotides inside of cells. Preferred syntheticoligonucleotides do not include a CGG quadmer or more than one CCG orCGG trimer at or near the 5′ and/or 3′ terminals and/or the consensusmitogenic CpG motif is not a palindrome. Prolonged immunostimulation canbe obtained using stabilized oligonucleotides, where the oligonucleotideincorporates a phosphate backbone modification. For example, themodification is a phosphorothioate or phosphorodithioate modification.More particularly, the phosphate backbone modification occurs at the 5′end of the nucleic acid for example, at the first two nucleotides of the5′ end of the nucleic acid. Further, the phosphate backbone modificationmay occur at the 3′ end of the nucleic acid for example, at the lastfive nucleotides of the 3′ end of the nucleic acid.

[0061] Preferably the immunostimulatory CpG DNA is in the range ofbetween 8 to 30 bases in size when it is an oligonucleotide.Alternatively, CpG dinucleotides can be produced on a large scale inplasmids, which after being administered to a subject are degraded intooligonucleotides. Preferred immunostimulatory nucleic acid molecules(e.g., for use in increasing the effectiveness of a vaccine or to treatan immune system deficiency by stimulating an antibody (i.e., humoral)response in a subject) have a relatively high stimulation index withregard to B cell, monocyte and/or natural killer cell responses (e.g.,cytokine, proliferative, lytic or other responses).

[0062] The nucleic acid sequences of the invention stimulate cytokineproduction in a subject for example. Cytokines include but are notlimited to IL-6, IL-12, IFN-γ, TNF-α and GM-CSF. Exemplary sequencesinclude: TCCATGTCGCTCCTGATGCT (SEQ ID NO: 37), TCCATGTCGTTCCTGATGCT (SEQID NO: 38), and TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 46).

[0063] The nucleic acid sequences of the invention are also useful forstimulating natural killer cell (NK) lytic activity in a subject such asa human. Specific, but non-limiting examples of such sequences include:TCGTCGTTGTCGTTGTCGTT (SEQ ID NO: 47), TCGTCGTTTTGTCGTTTTGTCGTT (SEQ IDNO: 46), TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 49), GCGTGCGTTGTCGTTGTCGTT(SEQ ID NO: 56), TGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 48),TGTCGTTGTCGTTGTCGTT (SEQ ID NO: 50) and TCGTCGTCGTCGTT (SEQ ID NO: 51).

[0064] The nucleic acid sequences of the invention are useful forstimulating B cell proliferation in a subject such as a human. Specific,but non-limiting examples of such sequences include:TCCTGTCGTTCCTTGTCGTT (SEQ ID NO: 52), TCCTGTCGTTTTTTGTCGTT (SEQ ID NO:53), TCGTCGCTGTCTGCCCTTCTT (SEQ ID NO: 54), TCGTCGCTGTTGTCGTTTCTT (SEQID NO: 55), TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 46),TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 49) and TGTCGTTGTCGTTGTCGTT (SEQ IDNO: 50).

[0065] In another aspect, the nucleic acid sequences of the inventionare useful as an adjuvant for use during antibody production in amammal. Specific, but non-limiting examples of such sequences include:TCCATGACGTTCCTGACGTT (SEQ ID NO: 10), GTCGTT (SEQ. ID. NO: 57), GTCGCT(SEQ. ID. NO. 58), TGTCGCT (SEQ. ID. NO: 101) and TGTCGTT (SEQ. ID. NO:102). Furthermore, the claimed nucleic acid sequences can beadministered to treat or prevent the symptoms of an asthmatic disorderby redirecting a subject's immune response from Th2 to Th1. An exemplarysequence includes TCCATGACGTTCCTGACGTT (SEQ ID NO: 10).

[0066] The stimulation index of a particular immunostimulatory CpG DNAcan be tested in various immune cell assays. Preferably, the stimulationindex of the immunostimulatory CpG DNA with regard to B-cellproliferation is at least about 5, preferably at least about 10, morepreferably at least about 15 and most preferably at least about 20 asdetermined by incorporation of ³H uridine in a murine B cell culture,which has been contacted with a 20 μM of ODN for 20 h at 37° C. and haspeen pulsed with 1 μCi of ³H uridine; and harvested and counted 4 hlater as described in detail in Example 1. For use in vivo, for exampleto treat an immune system deficiency by stimulating a cell-mediated(local) immune response in a subject, it is important that theimmunostimulatory CpG DNA be capable of effectively inducing cytokinesecretion by monocytic cells and/or Natural Killer (NK) cell lyticactivity.

[0067] Preferred immunostimulatory CpG nucleic acids should effect atleast about 500 pg/ml of TNF-α, 15 pg/ml IFN-γ, 70 pg/ml of GM-CSF 275pg/ml of IL-6, 200 pg/ml IL-12, depending on the therapeutic indication,as determined by the assays described in Example 12. Other preferredimmunostimulatory CpG DNAs should effect at least about 10%, morepreferably at least about 15% and most preferably at least about 20%YAC-1 cell specific lysis or at least about 30, more preferably at leastabout 35 and most preferably at least about 40% 2C11 cell specific lysisas determined by the assay described in detail in Example 4.

[0068] A “nucleic acid” or “DNA” means multiple nucleotides (i.e.,molecules comprising a sugar (e.g., ribose or deoxyribose) linked to aphosphate group and to an exchangeable organic base, which is either asubstituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U))or a substituted purine (e.g., adenine (A) or guanine (G)). As usedherein, the term refers to ribonucleotides as well asoligodeoxyribonucleotides. The term shall also include polynucleotides(i.e., a polynucleotide minus the phosphate) and any other organic basecontaining polymer. Nucleic acid molecules can be obtained from existingnucleic acid sources (e.g. genomic or cDNA), but are preferablysynthetic (e.g., produced by oligonucleotide synthesis).

[0069] A “nucleic acid delivery complex” shall mean a nucleic acidmolecule associated with (e.g., ionically or covalently bound to; orencapsulated within) a targeting means (e.g., a molecule that results inhigher affinity binding to target cell (e.g., B-cell and natural killer(NK) cell) surfaces and/or increased cellular uptake by target cells).Examples of nucleic acid delivery complexes include nucleic acidsassociated with: a sterol (e.g., a ligand recognized by target cellspecific receptor). Preferred complexes must be sufficiently stable invivo to prevent significant uncoupling prior to internalization by thetarget cell. However, the complex should be cleavable under appropriateconditions within the cell so that the nucleic acid is released in afunctional form.

[0070] “Palindromic sequences” shall mean an inverted repeat (i.e., asequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable offorming the usual Watson-Crick base pairs. In vivo, such sequences mayform double stranded structures.

[0071] A “stabilized nucleic acid molecule” shall mean a nucleic acidmolecule that is relatively resistant to in vivo degradation (e.g., viaan exo- or endo-nuclease). Stabilization can be a function of length orsecondary structure. Unmethylated CpG containing nucleic acid moleculesthat are tens to hundreds of kbs long are relatively resistant to invivo degradation. For shorter immunostimulatory nucleic acid molecules,secondary structure can stabilize and increase their effect. Forexample, if the 3′ end of a nucleic acid molecule hasself-complementarily to an upstream region, so that it can fold back andform a sort of stem loop structure, then the nucleic acid moleculebecomes stabilized and therefore exhibits more activity.

[0072] Preferred stabilized nucleic acid molecules of the instantinvention have a modified backbone. For use in immune stimulation,especially preferred stabilized nucleic acid molecules arephosphorothioate (i.e., at least one of the phosphate oxygens of thenucleic acid molecules is replaced by sulfur) or phosphorodithioatemodified nucleic acid molecules. More particularly, the phosphatebackbone modification occurs at the 5′ end of the nucleic acid forexample, at the first two nucleotides of the 5′ end of the nucleic acid.Further, the phosphate backbone modification may occur at the 3′ end ofthe nucleic acid for example, at the last five nucleotides of the 3′ endof the nucleic acid. In addition to stabilizing nucleic acid molecules,as reported further herein, phosphorothioate-modified nucleic acidmolecules (including phosphorodithioate-modified) can increase theextent of immune stimulation of the nucleic acid molecule, whichcontains an unmethylated CpG dinucleotide as shown herein. InternationalPatent Application Publication Number WO 95/26204 entitled “ImmuneStimulation By Phosphorothioate Oligonucleotide Analogs” also reports onthe non-sequence specific immunostimulatory effect of phosphorothioatemodified oligonucleotides. As reported herein, unmethylated CpGcontaining nucleic acid molecules having a phosphorothioate backbonehave been found to preferentially activate B-cell activity, whileunmethylated CpG containing nucleic acid molecules having aphosphodiester backbone have been found to preferentially activatemonocytic (macrophages, dendritic cells and monocytes) and NK cells.Phosphorothioate CpG oligonucleotides with preferred human motifs arealso strong activators of monocytic and NK cells.

[0073] Other stabilized nucleic acid molecules include: nonionic DNAanalogs, such as alkyl- and aryl-phosphonates (in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group),phosphodiester and alkylphosphotriesters, in which the charged oxygenmoiety is alkylated. Nucleic acid molecules which contain a diol, suchas tetraethylenglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

[0074] A “subject” shall mean a human or vertebrate animal including adog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, and mouse.

[0075] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Preferred vectors are those capable of autonomousreplication and expression of nucleic acids to which they are linked(e.g., an episome). Vectors capable of directing the expression of genesto which they are operatively linked are referred to herein as“expression vectors.” In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form, are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

[0076] Certain Unmethylated CpG Containing Nucleic Acids Have B CellStimulatory Activity As Shown In Vitro and In Vivo

[0077] In the course of investigating the lymphocyte stimulatory effectsof two antisense oligonucleotides specific for endogenous retroviralsequences, using protocols described in the attached Examples 1 and 2,it was surprisingly found that two out of twenty-four “controls”(including various scrambled, sense, and mismatch controls for a panelof “antisense” ODN) also mediated B cell activation and IgM secretion,while the other “controls” had no effect.

[0078] Two observations suggested that the mechanism of this B cellactivation by the “control” ODN may not involve antisense effects I)comparison of vertebrate DNA sequences listed in GenBank showed nogreater homology than that seen with non-stimulatory ODN and 2) the twocontrols showed no hybridization to Northern blots with 10 μg of spleenpoly A+ RNA. Resynthesis of these ODN on a different synthesizer orextensive purification by polyacrylainide gel electrophoresis or highpressure liquid chromatography gave identical stimulation, eliminatingthe possibility of an impurity. Similar stimulation was seen using Bcells from C3H/HeJ mice, eliminating the possibility thatlipopolysaccharide (LPS) contamination could account for the results.

[0079] The fact that two “control” ODN caused B cell activation similarto that of the two “antisense” ODN raised the possibility that all fourODN were stimulating B cells through some non-antisense mechanisminvolving a sequence motif that was absent in all of the othernonstimulatory control ODN. In comparing these sequences, it wasdiscovered that all of the four stimulatory ODN contained CpGdinucleotides that were in a different sequence context from thenonstimulatory control.

[0080] To determine whether the CpG motif present in the stimulatory ODNwas responsible for the observed stimulation, over 300 ODN ranging inlength from 5 to 42 bases that contained methylated, unmethylated, or noCpG dinucleotides in various sequence contexts were synthesized. TheseODNs, including the two original “controls” (ODN 1 and 2) and twooriginally synthesized as “antisense” (ODN 3D and 3M; Krieg, A. M. J.Immunol. 143:2448 (1989)), were then examined for in vitro effects onspleen cells (representative sequences are listed in Table I). SeveralODN that contained CpG dinucleotides induced B cell activation and IgMsecretion; the magnitude of this stimulation typically could beincreased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2aor 3D to 3Da and 3 Db). Stimulation did not appear to result from anantisense mechanism or impurity. ODN caused no detectable proliferationof γδ or other T cell populations.

[0081] Mitogenic ODN sequences uniformly became nonstimulatory if theCpG dinucleotide was mutated (Table 1; compare ODN 1 to 1a; 3D to 3Dc;3M to 3Ma; and 4 to 4a) or if the cytosine of the CpG dinucleotide wasreplaced by 5-methylcytosine (Table 1; ODN 1b, 2b, 3Dd, and 3 Mb).Partial methylation of CpG motifs caused a partial loss of stimulatoryeffect (compare 2a to 2c, Table 1). In contrast, methylation of othercytosines did not reduce ODN activity (ODN 1c, 2d, 3De and 3Mc). Thesedata confirmed that CpG motif is the essential element present in ODNthat activate B cells.

[0082] In the course of these studies, it became clear that the basesflanking the CpG dinucleotide played an important role in determiningthe murine B cell activation induced by an ODN. The optimal stimulatorymotif was determined to consist of a CpG flanked by two 5′ purines(preferably a GpA dinucleotide) and two 3′ pyrimidines (preferably a TpTor TpC dinucleotide). Mutations of ODN to bring the CpG motif closer tothis ideal improved the motif reduced stimulation (e.g., Table 1,compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutationsoutside the CpG motif did not reduce stimulation (e.g., Table I, compareODN to 1d; 3D to 3Dg; 3M to 3Me). For activation of human cells, thebest flanking bases are slightly different (See Table 5)).

[0083] Of those tested, ODNs shorter than 8 bases were non-stimulatory(e.g., Table 1, ODN 4e). Among the forty-eight 8 base ODN tested, ahighly stimulatory sequence was identified as TCAACGTT (SEQ. ID. NO: 90)(ODN4) which contains the self complementary “palindrome” AACGTT (SEQ.ID. NO: 105). In further optimizing this motif, it was found that ODNcontaining Gs at both ends showed increased stimulation, particularly ifthe ODN were rendered nuclease resistant by phosphorothioatemodification of the terminal internucleotide linkages. ODN 1585(GGGGTCAACGTTGAGGGGGG (SEQ ID NO: 12)), in which the first two and lastfive internucleotide linkages are phosphorothioate modified caused anaverage 25.4 fold increase in mouse spleen cell proliferation comparedto an average 3.2 fold increase in proliferation included by ODN 1638,which has the same sequence as ODN 1585 except that the 10 Gs at the twoends are replaced by 10 As. The effect of the G-rich ends is cis;addition of an ODN with poly G ends but no CpG motif to cells along with1638 gave no increased proliferation. For nucleic acid molecules longerthan 8 base pairs, non-palindromic motifs containing an unmethylated CpGwere found to be more immunostimulatory. TABLE 1 OligonucleotideStimulation of Mouse B Cells Stimulation Index′ CpG ODN Sequence (5′ to3′)† ³H IgM Production 1   (SEQ ID NO:89) GCTAGACGTTAGCGT 6.1 ± 0.8 17.9± 3.6  1a  (SEQ. ID NO:4) ......T........ 1.2 ± 0.2 1.7 ± 0.5 1b  (SEQID NO:13) ......Z........ 1.2 ± 0.1 1.8 ± 0.0 1c  (SEQ ID NO:14)............Z.. 10.3 ± 4.4  9.5 ± 1.8 1d  (SEQ ID NO:16) ..AT......GAGC.13.0 ± 2.3  18.3 ± 7.5  2   (SEQ ID NO:1) ATGGAAGGTCCAGCGTTCTC 2.9 ± 0.213.6 ± 2.0  2a  (SEQ ID NO:15) ..C..CTC..G......... 7.7 ± 0.8 24.2± 3.2  2b  (SEQ ID NO:16) ..Z..CTC.ZG..Z...... 1.6 ± 0.5 2.8 ± 2.2 2c (SEQ ID NO:17) ..Z..CTC..G......... 3.1 ± 0.6 7.3 ± 1.4 2d  (SEQ IDNO:18) ..C..CTC..G......Z.. 7.4 ± 1.4 27.7 ± 5.4  2e  (SEQ ID NO:19)............A....... 5.6 ± 2.0 ND 3D  (SEQ ID NO:20)GAGAACGCTGGACCTTCCAT 4.9 ± 0.5 19.9 ± 3.6  3Da (SEQ ID NO:21).........C.......... 6.6 ± 1.5 33.9 ± 6.8  3Db (SEQ ID NO:22).........C.......G.. 10.1 ± 2.8  25.4 ± 0.8  3Dc (SEQ ID NO:23)...C.A.............. 1.0 ± 0.1 1.2 ± 0.5 3Dd (SEQ ID NO:24).....Z.............. 1.2 ± 0.2 1.0 ± 0.4 3De (SEQ ID NO:25).............Z...... 4.4 ± 1.2 18.8 ± 4.4  3Df (SEQ ID NO:26).......A............ 1.6 ± 0.1 7.7 ± 0.4 3Dg (SEQ ID NO:27).........CC.G.ACTG.. 6.1 ± 1.5 18.6 ± 1.5  3M  (SEQ ID NO:28)TCCATGTCGGTCCTGATGCT 4.1 ± 0.2 23.2 ± 4.9  3Ma (SEQ ID NO:29)......CT............ 0.9 ± 0.1 1.8 ± 0.5 3Mb (SEQ ID NO:30).......Z............ 1.3 ± 0.3 1.5 ± 0.6 3Mc (SEQ ID NO:31)...........Z........ 5.4 ± 1.5 8.5 ± 2.6 3Md (SEQ ID NO:37)......A..T.......... 17.2 ± 9.4  ND 3Me (SEQ ID NO:32)...............C..A. 3.6 ± 0.2 14.2 ± 5.2  4   (SEQ ID NO:90) TCAACGTT6.1 ± 1.4 19.2 ± 5.2  4a  (SEQ ID NO:91) ....GC.. 1.1 ± 0.2 1.5 ± 1.14b  (SEQ ID NO:92) ...GCGC. 4.5 ± 0.2 9.6 ± 3.4 4c  (SEQ ID NO:93)...TCGA. 2.7 ± 1.0 ND 4d  (SEQ ID NO:94) ..TT..AA 1.3 ± 0.2 ND 4e  (SEQID NO:106) -....... 1.3 ± 0.2 1.1 ± 0.5 4f  (SEQ ID NO:95) C....... 3.9± 1.4 ND 4g  (SEQ ID NO:107) --......CT 1.4 ± 0.3 ND 4h  (SEQ ID NO:96).......C 1.2 ± 0.2 ND LPS 7.8 ± 2.5 4.8 ± 1.0

[0084] Other octamer ODN containing a 6 base palindrome with a TpCdinucleotide at the 5′ end were also active (e.g., Table 1, ODN 4b, 4c).Other dinucleotides at the 5′ end gave reduced stimulation (e.g., ODN4f, all sixteen possible dinucleotides were tested). The presence of a3′ dinucleotide was insufficient to compensate for the lack of a 5′dinucleotide (e.g., Table 1, ODN 4g). Disruption of the palindromeeliminated stimulation in octamer ODN (e.g., Table 1, ODN 4h), butpalindromes were not required in longer ODN.

[0085] The kinetics of lymphocyte activation were investigated usingmouse spleen cells. When the cells were pulsed at the same time as ODNaddition and harvested just four hours later, there was already atwo-fold increase in 3H uridine incorporation. Stimulation peaked at12-48 hours and then decreased. After 24 hours, no intact ODN weredetected, perhaps accounting for the subsequent fall in stimulation whenpurified B cells with or without anti-IgM (at a submitogenic dose) werecultured with CpG ODN, proliferation was found to synergisticallyincrease about 10-fold by the two mitogens in combination after 48hours. The magnitude of stimulation was concentration dependent andconsistently exceeded that of LPS under optimal conditions for both.Oligonucleotides containing a nuclease resistant phosphorothioatebackbone were approximately two hundred times mor potent than unmodifiedoligonucleotides.

[0086] Cell cycle analysis was used to determine the proportion of Bcells activated by CpG-ODN. CpG-ODN induced cycling in more than 95% ofB cells. Splenic B lymphocytes sorted by flow cytometry intoCD23−(marginal zone) and CD23+(follicular) subpopulations were equallyresponsive to ODN-induced stimulation, as were both resting andactivated populations of B cells isolated by fractionation over Percollgradients. These studies demonstrated that CpG-ODN induced essentiallyall B cells to enter the cell cycle.

[0087] Immunostimulatory Nucleic Acid Molecules Block Murine B CellApoptosis

[0088] Certain B cell lines, such as WEHI-231, are induced to undergogrowth arrest and/or apoptosis in response to crosslinking of theirantigen receptor by anti-IgM (Jakway, J. P. et al., “Growth regulationof the B lymphoma cell line WEHI-231 by anti-immunoglobulin,lipopolysaccharide and other bacterial products” J. Immunol. 137: 2225(1986); Tsubata, T., J. Wu and T. Honjo: B-cell apoptosis induced ybantigen receptor crosslinking is blocked by a T-cell signal throughCD40.” Nature 365: 645 (1993)). WEHI-231 cells are rescued from thisgrowth arrest by certain stimuli such as LPS and by the CD40 ligand. ODNcontaining the CpG motif were also found to protect WEHI-231 fromanti-IgM induced growth arrest, indicating that accessory cellpopulations are not required for the effect. Subsequent work indicatesthat CpG ODN induce Bcl-x and myc expression, which may account for theprotection from apoptosis. Also, CpG nucleic acids have been found toblock apoptosis in human cells. This inhibition of apoptosis isimportant, since it should enhance and prolong immune activation by CpGDNA.

[0089] Identification of the Optimal CpG Motif for Induction of MurineIL-6 and IgM Secretion and B Cell Proliferation.

[0090] To evaluate whether the optimal B cell stimulatory CpG motif wasidentical with the optimal CpG motif for IL-6 secretion, a panel of ODNin which the bases flanking the CpG dinucleotide were progressivelysubstituted was studied. This ODN panel was analyzed for effects on Bcell proliferation, Ig production, and IL-6 secretion, using bothsplenic B cells and CH12.LX cells. As shown in Table 2, the optimalstimulatory motif contains an unmethylated CpG flanked by two 5′ purinesand two 3′ pyrimidines. Generally a mutation of either 5′ purines to Cwere especially deleterious, but changes in 5′ purines to T or 3′pyrimidines to purines had less marked effects. Based on analyses ofthese and scores of other ODN, it was determined that the optimal CpGmotif for induction of IL-6 secretion is TGACGTT (SEQ. ID. NO: 108),which is identical with the optimal mitogenic and IgM-inducing CpG motif(Table 2). This motif was more stimulatory than any of the palindromecontaining sequences studied (1639, 1707 and 1708).

[0091] Induction of Murine Cytokine Secretion by CpG Motifs in BacterialDNA or Oligonucleotides.

[0092] As described in Example 9, the amount of IL-6 secreted by spleencells after CpG DNA stimulation was measured by ELISA. T cell depletedspleen cell cultures rather than whole spleen cells were used for invitro studies following preliminary studies showing that T cellscontribute little or nothing to the IL-6 produced by CpG DNA-stimulatedspleen cells. As shown in Table 3, IL-6 production was markedlyincreased in cells cultured with E. coli DNA but not in cells culturedwith calf thymus DNA. To confirm that the increased IL-6 productionobserved with E. coli DNA was not de to contamination by other bacterialproducts, the DNA was digested with DNAse prior to analysis. DNAsepretreatment abolished IL-6 production induced by E. coli DNA (Table 3).In addition, spleen cells from LPS-nonresponsive C2H/HeJ mouse producedsimilar levels of IL-6 in response to bacterial DNA. To analyze whetherthe IL-6 secretion induced by E. coli DNA was mediated by theunmethylated CpG dinucleotides in bacterial DNA, methylated E. coli DNAand a panel of synthetic ODN were examined. As shown in Table 3, CpG ODNsignificantly induced IL-6 secretion (ODN 5a, 5b, 5c) while CpGmethylated E. coli DNA, or ODN containing methylated CpG (ODN 5f) or noCpG (ODN 5d) did not. Changes at sites other than CpG dinucleotides (ODN5b) or methylation of other cytosines (ODN 5g) did not reduce the effectof CpG ODN. Methylation of a single CpG in an ODN with three CpGsresulted in a partial reduction in the stimulation (compare ODN 5c to5e; Table 3). TABLE 3 Induction of Murine IL-6 secretion by CpG motifsin bacterial DNA or oligonucleotides Treatment IL-6 (pg/ml) calf thymusDNA ≦10 calf thymus DNA + DNase ≦10 E. coli DNA 1169.5 ± 94.1  E. coliDNA + DNase ≦10 CpG methylated E. coli DNA ≦10 LPS  280.1 ± 17.1  Media(no DNA) ≦10 ODN5a SEQ. ID. No:115 TGGACTCTCCAGCGTTCTC 1096.4 ± 372.0   5b SEQ. ID. No:19 .....AGG....A....... 1124.5 ± 126.2    5c SEQ. ID.No:15 ..C.......G......... 1783.0 ± 189.5    5d SEQ. ID. No:124.... AGG..C..T...... ≦10    5e SEQ. ID. No:116 ..C.......G..Z...... 851.1 ± 114.4    5f SEQ. ID. No:16 ..Z......ZG..Z...... ≦10    5g SEQ.ID. No:18 ..C.......G......Z.. 1862.3 + 87.26

[0093] CpG Motifs can be used as an Artificial Adjuvant.

[0094] Nonspecific simulators of the immune response are known asadjuvants. The use of adjuvants is essential to induce a strong antibodyresponse to soluble antigens (Harlow and Lan, Antibodies: A Laboratorymanual, Cold Spring Harbor, N.Y. Current Edition; hereby incorporated byreference). The overall effect of adjuvants is dramatic and theirimportance cannot be overemphasized. The action of an adjuvant allowsmuch smaller doses of antigen to be used and generates antibodyresponses that are more persistent. The nonspecific activation of theimmune response often can spell the difference between success andfailure in obtaining an immune response. Adjuvants should be used forfirst injections unless there is some very specific reason to avoidthis. Most adjuvants incorporate two components. One component isdesigned to protect the antigen from rapid catabolism (e.g., liposomesor synthetic surfactants (Hunter et al. 1981)). Liposomes are onlyeffective when the immunogen is incorporated into the outer lipid layer;entrapped molecules are not seen by the immune system. The othercomponent is a substance that will stimulate the immune responsenonspecifically. These substances act by raising the level oflymphokines. Lymphokines stimulate the activity of antigen-processingcells directly and cause a local inflammatory reaction at the site ofinjection. Early work relied entirely on heat-killed bacteria (Dienes1936) or lipopolysaccharide (LPS) (Johnson et al. 1956). LPS isreasonably toxic, and, through analysis of its structural components,most of its properties as an adjuvant have been shown to be in a portionknown as lipid A. Lipid A is available in a number of synthetic andnatural forms that are much less toxic than LPS, but still retains mostof the better adjuvant properties of parental LPS molecule. Lipid Acompounds are often delivered using liposomes.

[0095] Recently an intense drive to find potent adjuvants with moreacceptable side effects has led to the production of new syntheticadjuvants. The present invention provides the sequence 1826TCCATGACGTTCCTGACGTT (SEQ ID NO: 10), which is an adjuvant including CpGcontaining nucleic acids. The sequence is a strong immune activatingsequence and is a superb adjuvant, with efficacy comparable or superiorto complete Freund's, but without apparent toxicity.

[0096] Titration of Induction of Murine IL-6 Secretion by CpG motifsBacterial DNA and CpG ODN induced IL-6 production in T cell depletedmurine spleen cells in a dose-dependent manner, but vertebrate DNA andnon-CpG ODN did not (FIG. 1). IL-6 production plateaued at approximately50 μg/ml of bacterial DNA or 40 μM of CpG O-ODN. The maximum levels ofIL-6 induced by bacterial DNA and CpG ODN were 1-1.5 ng/ml and 2-4 ng/mlrespectively. These levels were significantly greater than thoseseen-after stimulation by LPS (0.35 ng/ml) (FIG. 1A). To evaluatewhether CpG ODN with a nuclease-resistant DNA backbone would also induceIL-6 production, S-ODN were added to T cell depleted murine spleencells. CpG S-ODN also induced IL-6 production in a dose-dependent mannerto approximately the same level as CpG O-ODN while non-CpG S-ODN failedto induce IL-6 (FIG. 1C). CpG S-ODN at a concentration of 0.05 μM couldinduce maximal IL-6 production in these cells. This result indicatedthat the nuclease-resistant DNA backbone modification retains thesequence specific ability of CpG DNA to induce IL-6 secretion and thatCpG S-ODN are more than 80-fold more potent than CpG O-ODN in this assaysystem.

[0097] Induction of Murine IL-6 secretion by CpG DNA In Vivo

[0098] To evaluate the ability of bacterial DNA and CpG S-ODN to induceIL-6 secretion in vivo, BALB/c mice were injected iv. with 100 μg of E.coli DNA, calf thymus DNA, or CpG or non-stimulatory S-ODN and bled 2 hrafter stimulation. The level of IL-6 in the sera from the E. coli DNAinjected group was approximately 13 ng/ml while IL-6 was not detected inthe sera from calf thymus DNA or PBS injected groups (Table 4). CpGS-ODN also induced IL-6 secretion in vivo. The IL-6 level in the serafrom CpG S-ODN injected groups was approximately 20 ng/ml. In contrast,IL-6 was not detected in the sera from non-stimulatory S-ODN stimulatedgroup (Table 4). TABLE 4 Secretion of Murine IL-6 induced by CpG DNAstimulation in vivo. Stimulant IL-6 (pg/ml) PBS <50 E. coli DNA 13858± 3143 Calf Thymus DNA <50 CpG S-ODN 20715 ± 606 (5′GCATGACGTTGAGCT3′)(SEQ. ID. No:48) non-CpG S-ODN <50 (5′GCTAGATGTTAGCGT3′) (SEQ. ID.No:49) # non-stimulatory S-ODN is 5′GCTAGATGTTAGCGT3′ (SEQ. ID. No:4).

[0099] Kinetics of Murine IL-6 Secretion after Stimulation by CpG MotifsIn Vivo

[0100] To evaluate the kinetics of induction of IL-6 secretion by CpGDNA n vivo, BALB/c mice were injected iv. with CpG or control non-CpGS-ODN. Serum IL-6 levels were significantly increased within 1 hr andpeaked at 2 hr to a level of approximately 9 ng/ml in the CpG S-ODNinjected group (FIG. 2). IL-6 protein in sera rapidly decreased after 4hr and returned to basal level by 12 hr after stimulation. In contrastto CpG DNA stimulated groups, no significant increase of IL-6 wasobserved in the sera from the non-stimulatory S-ODN or PBS injectedgroups (FIG. 2).

[0101] Tissue Distribution and Kinetics of IL-6, mRNA Expression Inducedby CpG Motifs In Vivo

[0102] As shown in FIG. 2, the level of serum IL-6 increased rapidlyafter CpG DNA stimulation. To investigate the possible tissue origin ofthis serum IL-6, and the kinetics of IL-6 gene expression in vivo afterCpG DNA stimulation, BALB/c mice were injected iv with CpG or non-CpGS-ODN and RNA was extracted from liver, spleen, thymus, and bone marrowat various time points after stimulation. As shown in FIG. 3A, the levelof IL-6 mRNA in liver, spleen, and thymus was increased within 30 min.after injection of CpG S-ODN. The liver IL-6 mRNA peaked at 2 hrpost-injection and rapidly decreased and reached basal level 8 hr afterstimulation (FIG. 3A). Splenic IL-6 mRNA peaked at 2 hr afterstimulation and then gradually decreased (FIG. 3A). Thymus IL-6 mRNApeaked at 1 hr post-injection and then gradually decreased (FIG. 3A).IL-6 mRNA was significantly increased in bone marrow within 1 hr afterCpG S-ODN injection but then returned to basal level. In response to CpGS-ODN, liver, spleen and thymus showed more substantial increases inIL-6 mRNA expression than the bone marrow.

[0103] Patterns of Murine Cytokine Expression Induced by CpG DNA

[0104] In vivo or in whole spleen cells, no significant increase in theprotein levels of the following interleukins: IL-2, IL-3, IL-4, IL-5, orIL-10 was detected within the first six hours (Klinman, D. M. et al.,(1996) Proc. Natl. Acad. Sci. USA 93:2879-2883). However, the level ofTNF-α is increased within 30 minutes and the level of IL-6 increasedstrikingly within 2 hours in the serum of mice injected with CpG ODN.Increased expression of IL-12 and interferon gamma (IFN-γ) mRNA byspleen cells was also detected within the first two hours. dots indicateTABLE 5 Induction of human PBMC cytokine secrtetion by CpG oligos ODNSequence (5′-3′) IL-6¹ TNF-α¹ IFN-γ¹ GM-CSF IL-12  512TCCATGTCGGTCCTGATGCT 500 140 15.6 70 250 SEQ ID NO:28  1637......C............ 550 16 7.8 15.6 35 SEQ ID NO:29  1615......G............. 600 145 7.8 45 250 SEQ ID NO:101 1614......A............. 550 31 0 50 250 SEQ ID NO:102 1636.........A.......... 325 250 35 40 0 SEQ ID NO:103 1634.........C.......... 300 400 40 85 200 SEQ ID NO:104 1619.........T.......... 275 450 200 80 >500 SEQ ID NO:105 1618......A..T.......... 300 60 15.6 15.6 62 SEQ ID NO:7   1639.....AA..T.......... 625 220 15.6 40 60 SEQ ID NO:3   1707......A..TC......... 300 70 17 0 0 SEQ ID NO:88  1708.....CA..TG......... 270 10 17 0 0 SEQ ID NO:106

[0105] CpG DNA Induces Cytokine Secretion by Human PBMC SpecificallyMonocytes

[0106] The same panels of ODN used for studying mouse cytokineexpression were used to determine whether human cells also are inducedby CpG motifs to express cytokine (or proliferate), and to identify theCpG motif(s) responsible. Oligonucleotide 1619 (GTCGTT; residues of 6-11of SEQ. ID. NO: 105) was the best inducer of TNF-α and IFN-γ secretion,and was closely followed by a nearly identical motif in oligonucleotide1634 (GTCGCT; residues 6-11 of SEQ. ID. NO: 104) (Table 5). The motifsin oligodeoxynucleotides 1637 and 1614 (GCCGGT; residues 6-11 of SEQ.ID. NO: 29) and (GACGGT; residues 6-11 of SEQ. ID. NO: 102) led tostrong IL-6 secretion with relatively little induction of othercytokines. Thus, it appears that human lymphocytes, like murinelymphocytes, secrete cytokines differentially in response to CpGdinucleotides, depending on the surrounding bases. Moreover, the motifsthat stimulate murine cells best differ from those that are mosteffective with human cells. Certain CpG oligodeoxynucleotides are poorat activating human cells (oligodeoxynucleotides 1707, 1708, whichcontain the palindrome forming sequences GACGTC residues 6-11 of SEQ.ID. NO: 88 and CACGTG residues 6-11 of SEQ. ID. NO: 106, respectively).

[0107] The cells responding to the DNA appear to be monocytes, since thecytokine secretion is abolished by treatment of the cells withL-leucyl-L-leucine methyl ester (L-LME), which is selectively toxic tomonocytes (but also to cytotoxic T lymphocytes and NK cells), and doesnot affect B cell Ig secretion (Table 6). The cells surviving L-LMEtreatment had >95% viability by trypan blue exclusion, indicating thatthe lack of a cytokine response among these cells did not simply reflecta nonspecific death of all cell types. Cytokine secretion in response toE. coli (EC) DNA requires unmethylated CpG motifs, since it is abolishedby methylation of the EC DNA (next to the bottom row, Table 6). LPScontamination of the DNA cannot explain the results since the level ofcontamination was identical in the native and methylated DNA, and sinceaddition of twice the highest amount of contaminating LPS had no effect(not shown). TABLE 6 CpG DNA induces cytokine secretion by human PBMCTNF-α IL-6 IFN-γ RANTES DNA (pg/ml)¹ (pg/ml) (pg/ml) (pg/ml) EC DNA (50μ/ml) 900 12,000 700 1560 EC DNA (5 μ/ml) 850 11,000 400 750 EC DNA (0.5μ/ml) 500 ND 200 0 EC DNA (0.05 μ/ml) 62.5 10,000 15.6 0 EC DNA (50μ/ml) + L-LME² 0 ND ND ND EC DNA (10 μ/ml) Methyl³ 0 5 ND ND CT DNA (50μ/ml) 0 600 0 0

[0108] The loss of cytokine production in the PBMC treated with L-LMEsuggested that monocytes may be responsible for cytokine production inresponse to CpG DNA. To test this hypothesis more directly, the effectsof CpG DNA on highly purified human monocytes and macrophages wastested. As hypothesized, CpG DNA directly activated production of thecytokines IL-6, GM-CSF, and TNF-α by human macrophages, whereas non-CpGDNA did not (Table 7). TABLE 7 CpG DNA induces cytokine expression inpurified human macrophages IL-6 (pg/ml) GM-CSF (pg/ml) TNF-α (pg/ml)Cells alone   0 0   0 CT DNA (50 μg/ml)   0 0   0 EC DNA (50 μg/ml) 200015.6 1000

[0109] Biological Role of IL-6 in Inducing Murine IgM Production inResponse to CpG Motifs

[0110] The kinetic studies described above revealed that induction ofIL-6 secretion, which occurs within 1 hr post CpG stimulation, precedesIgM secretion. Since the optimal CpG motif for ODN inducing secretion ofIL-6 is the same as that for IgM (Table 2), whether the CpG motifsindependently induce IgM and IL-6 production or whether the IgMproduction is dependent on prior IL-6 secretion was examined. Theaddition of neutralizing anti-IL-6 antibodies inhibited in vitro IgMproduction mediated by CpG ODN in a dose-dependent manner but a controlantibody did not (FIG. 4A). In contrast, anti-IL-6 addition did notaffect either the basal level or the CpG-induced B cell proliferation(FIG. 4B).

[0111] Increased Transcriptional Activity of the IL-6 Promoter inResponse to CpG DNA

[0112] The increased level of IL-6 mRNA and protein after CpG DNAstimulation could result from transcriptional or post-transcriptionalregulation. To determine if the transcriptional activity of the IL-6promoter was unregulated in B cells cultured with CpG ODN, a murine Bcell line, WEHI-231, which produces IL-6 in response to CpG DNA, wastransfected with an IL-6 promoter-CAT construct (pIL-6/CAT) (Pottrats,S. T. et al., 17B-estradiol) inhibits expression of humaninterleukin-6-promoter-reporter constructs by a receptor-dependentmechanism. J. Clin. Invest. 93:944). CAT assays were performed afterstimulation with various concentrations of CpG or non-CpG ODN. As shownin FIG. 5, CpG ODN induced increased CAT activity in dose-dependentmanner while non-CPG ODN failed to induce CAT activity. This confirmsthat CpG induces the transcriptional activity of the IL-6 promoter.

[0113] Dependence of B Cell Activation by CpG ODN on the Number of 5′and 3′ Phosphorothioate Internucleotide Linkages

[0114] To determine whether partial sulfur modification of the ODNbackbone would be sufficient to enhance B cell activation, the effectsof a series of ODN with the same sequence, but with differing numbers ofS internucleotide linkages at the 5′ end of ODN were required to provideoptimal protection of the ODN from degradation by intracellular exo- andendo-nucleases. Only chimeric ODN containing two 5′phosphorothioate-modified linkages, and a variable number of 3′ modifiedlinkages were therefore examined.

[0115] The lymphocyte stimulating effects of these ODN were tested atthree concentrations (3.3, 10, and 30 μM) by measuring the total levelsof RNA synthesis (by ³H uridine incorporation) or DNA synthesis (by ³Hthymidine incorporation) in treated spleen cell cultures (Example 10).O-ODN (0/0 phosphorothioate modifications) bearing a CpG motif caused nospleen cell stimulation unless added to the cultures at concentrationsof at least 10 μM (Example 10). However, when this sequence was modifiedwith two S linkages at the 5′ end and at least three S linkages at the3′ end, significant stimulation was seen at a dose of 3.3 μM. At thislow dose, the level of stimulation showed a progressive increase as thenumber of 3′ modified bases was increased, until this reached orexceeded six, at which point the stimulation index began to decline. Ingeneral, the optimal number of 3′ S linkages for spleen cell stimulationwas five. Of all three concentrations tested in these experiments, theS-ODN was less stimulatory than the optimal chimeric compounds.

[0116] Dependent of GpG-mediated Lymphocyte Activation on the Type ofBackbone Modification

[0117] Phosphorothioate modified ODN (S-ODN) are far more nucleaseresistant than phosphodiester modified ODN (O-ODN). Thus, the increasedimmune stimulation caused by S-ODN and S-O-ODN (i.e., chimericphosphorothioate ODN in which the central linkages are phosphodiester,but the two 5′ and five 3′ linkages are phosphorothioate modified)compared to O-ODN may result from the nuclease resistance of the former.To determine the role of ODN nuclease resistance in immune stimulationby CpG ODN, the stimulatory effects of chimeric ODN in which the 5′ and3′ ends were rendered nuclease resistant with either methylphosphonate(MP-), methylphosphorothioate (MPS-), phosphorothioate (S-), orphosphorodithioate (S₂-) internucleotide linkages were tested (Example10). These studies showed that despite their nuclease resistance,MP-O-ODN were actually less immune stimulatory than O-ODN. However,combining the MP and S modifications by replacing both nonbridging Omolecules with 5′ and 3′ MPS internucleotide linkages restored immunestimulation to a slightly higher level than that triggered by O-ODN.

[0118] S-O-ODN were far more stimulatory than O-ODN, and were even morestimulatory than S-ODN, at least at concentrations above 3.3 μM. Atconcentrations below 3 μM, the S-ODN with the 3M sequence was morepotent than the corresponding S-O-ODN, while the S-ODN with the 3Dsequence was less potent than the corresponding S-O-ODN (Example 10). Incomparing the stimulatory CpG motifs of these two sequences, it wasnoted that the 3D sequence is a perfect match for the stimulatory motifin that the CpG is flanked by two 5′ purines and two 3′ pyrimidines.However, the bases immediately flanking the CpG in ODN 3D are notoptimal; it has a 5′ pyrimidine and a 3′ purine. Based on furthertesting, it was found that the sequence requirement for immunestimulation is more stringent for S-ODN than for S-O- or O-ODN. S-ODNwith poor matches to the optimal CpG motif cause little or no lymphocyteactivation (e.g., Sequence 3D). However, S-ODN with good matches to themotif, most critically at the positions immediately flanking the CpG,are more potent than the corresponding S-O-ODN (e.g., Sequence 3M,Sequences 4 and 6), even though at higher concentrations (greater than 3μM) the peak effect from the S-O-ODN is greater (Example 10).

[0119] S₂O-ODN were remarkably stimulatory, and caused substantiallygreater lymphocyte activation than the corresponding S-ODN or S-O-ODN atevery tested concentration.

[0120] The increased B cell stimulation seen with CpG ODN bearing S orS₂ substitutions could result from any or all of the following effects:nuclease resistance, increased cellular uptake, increased proteinbinding, and altered intracellular localization. However, nucleaseresistance cannot be the only explanation, since the MP-O-ODN wereactually less stimulatory than the O-ODN with CpG motifs. Prior studieshave shown that ODN uptake by lymphocytes is markedly affected by thebackbone chemistry (Zhao, et al. (1993) Comparison of cellular bindingand uptake of antisense phosphodiester, phosphorothioate, and mixedphosphorothioate and methylphosphonate oligonucleotides. (AntisenseResearch and Development 3, 53-66; Zhao et al., (1994) Stage specificoligonucleotide uptake in murine bone marrow B cell precursors. Blood84, 3660-3666). The highest cell membrane binding and uptake was seenwith S-ODN, followed by S-O-ODN, O-ODN, and MP-ODN. This differentialuptake correlates with the degree of immune stimulation.

[0121] Unmethylated CpG Containing Oligos Have NK Cell StimulatoryActivity

[0122] Experiments were conducted to determine whether CpG containingoligonucleotides stimulated the activity of natural killer (NK) cells inaddition to B cells. As shown in Table 8, a marked induction of NKactivity among spleen cells cultured with CpG ODN 1 and 3Dd wasobserved. In contrast, there was relatively on induction in effectorsthat had been treated with non-CpG control ODN. TABLE 8 Induction Of NKActivity By CpG Oligodeoxynucleotides (ODN) % YAC-1 Spe- % 2C11 Spe-cific Lysis* cific Lysis Effector: Target Effector: Target ODN 50:1100:1 50:1 100:1 None −1.1 −1.4 15.3 16.6 I (SEQ ID NO:13) 16.1 24.538.7 47.2 3Dd (SEQ ID NO:27) 17.1 27.0 37.0 40.0 Non-CpG ODN −1.6 −1.714.8 15.4

[0123] Induction of NK Activity by DNA Containing CpG Motifs, but not bynon-CpG DNA.

[0124] Bacterial DNA cultured for 18 hrs at 37° C. and then assayed forkilling of K562 (human) or Yac-1 (mouse) target cells induced NK lyticactivity in both mouse spleen cells depleted of B cells and human PBMC,but vertebrate DNA may be a consequence of its increased level ofunmethylated CpG dinucleotides, the activating properties of more than50 synthetic ODN containing unmethylated, methylated, or no CpGdinucleotides was tested. The results, summarized in Table 9,demonstrate that synthetic ODN can stimulate significant NK activity, aslong as they contain at least one unmethylated CpG dinucleotide. Nodifference was observed in the stimulatory effects of ODN in which theCpG was within a palindrome (such as ODN 1585, which contains thepalindrome AACGTT; SEQ. ID. NO: 105) from those ODN without palindromes(such as 1613 ro 1619), with the caveat that optimal stimulation wasgenerally seem with ODN in which the CpG was flanked by two 5′ purinesor a 5′ GpT dinucleotide and two 3′ pyrimidines. Kinetic experimentsdemonstrated that NK activity peaked around 18 hrs. after addition ofthe ODN. The data indicates that the murine NK response is dependent onthe prior activation of monocytes by CpG DNA, leading to the productionof IL-12, TNF-α, and IFN-α/β (Example 11). TABLE 9 Induction of NKActivity by DNA Containing CpG Motifs but not by Non-CpG DNA LU/10⁶ DNAor Cytokine Added Mouse Cells Human Cells Expt. 1 None 0.00 0.00 IL-216.68 15.82 E. Coli. DNA 7.23 5.05 Calf thymus DNA 0.00 0.00 Expt. 2None 0.00 3.28 1585 ggGGTCAACGTTGAGggggg (SEQ ID No.12) 7.38 17.98 1629-------gtc------- (SEQ ID No.41) 0.00 4.4 Expt. 3 None 0.00 1613GCTAGACGTTAGTGT (SEQ ID No.42) 5.22 1769 -------Z------- (SEQ ID No.52)0.02 ND 1619 TCCATGTCGTTCCTGATGCT (SEQ ID No.38) 3.35 1765-------Z------- (SEQ ID No.44) 0.11

[0125] From all of these studies, a more complete understanding of theimmune effects of CpG DNA has been developed, which is summarized inFIG. 6.

[0126] Immune activation by CpG motifs may depend on bases flanking theCpG, and the number of spacing of the CpGs present within an ODN.Although a single CpG in an ideal base context can be a very strong anduseful immune activator, superior effects can be seen with ODNcontaining several CpGs with the appropriate spacing and flanking bases.For activation of murine B cells, the optimal CpG motif is TGACGTT (SEQ.ID. NO: 108); residues 11-17 of Seq. ID. No 70.

[0127] The following studies where conducted to identify optimal ODNsequences for stimulation of human cells by examining the effects ofchanging the number, spacing, and flanking bases of CpG dinucleotides.

[0128] Identification of Phosphorothioate ODN with Optimal CpG Motifsfor Activation of Human NK Cells.

[0129] To have clinical utility, ODN must be administered to a subjectin a form that protects them against nuclease degradation. Methods toaccomplish this with phosphodiester ODN are well known in the art andinclude encapsulation in lipids or delivery systems such asnanoparticles. This protection can also be achieved using chemicalsubstitutions to the DNA such as modified DNA backbones including thosein which the internucleotide linkages are nuclease resistant. Somemodifications may confer additional desirable properties such asincreasing cellular uptake. For example, the phosphodiester linkage canbe modified via replacement of one of the nonbridging oxygen atoms witha sulfur, which constitutes phosphorothioate DNA. Phosphorothioate ODNhave enhanced cellular uptake (Krieg et al., Antisense Res. Dev. 6:133,1996.) and improved B cell stimulation if they also have a CpG motif.Since NK activation correlates strongly with in vivo adjuvant effects,the identification of phosphorothioate ODN that will activate human NKcells is very important.

[0130] The effects of different phosphorothioate ODNs—containing CpGdinucleotides in various base contexts—on human NK activation (Table 10)were examined. ODN 1840, which contained 2 copies of the TGTCGTT (SEQ.ID. NO: 102) residues 14-20 of SEQ. ID. NO: 47 motif, had significant NKlytic activity (Table 10). To further identify additional ODNs optimalfor NK activation, approximately one hundred ODN containing differentnumbers and spacing of CpG motifs, were tested with ODN1982 serving as acontrol. The results are shown in Table 11.

[0131] Effective ODNs began with a TC or TG at the 5′ end, however, thisrequirement was not mandatory. ODNs with internal CpG motifs (e.g., ODN1840) are generally less potent stimulators than those in which a GTCGCT(SEQ. ID. NO: 58) motif immediately follows the 5′ TC (e.g., ODN 1967and 1968). ODN 1968, which has a second GTCGTT (SEQ. ID. NO: 57) motifin its 3′ half, was consistently more stimulatory than ODN 1967, whichlacks this second motif. ODN 1967, however, was slightly more potentthan ODN 1968 in experiments 1 and 3, but not in experiment 2. ODN 2005,which has a third GTCGTT (SEQ. ID. NO: 57) motif, inducing slightlyhigher NK activity on average than 1968. However, ODN 2006, in which thespacing between the GTCGTT (SEQ. ID. NO: 57) motifs was increased by theaddition of two Ts between each motif, was superior to ODN 2005 and toODN 2007, in which only one of the motifs had the additional of thespacing two Ts. The minimal acceptable spacing between CpG motifs is onenucleotide as long as the ODN has two pyrimidines (preferably T) at the3′ end (e.g., ODN 2015). Surprisingly, joining two GTCGTT (SEQ. ID. NO:57) motifs end to end with a 5′ T also created a reasonably stronginducer of NK activity (e.g., ODN 2016). The choice of thymine (T)separating consecutive CpG dinucleotides is not absolute, since ODN 2002induced appreciable NK activation despite the fact that adenine (A)separated its CpGs (i.e., CGACGTT; SEQ. ID. NO: 113). It should also benoted that ODNs containing no CpG (e.g., ODN 1982), runs of CpGs, orCpGs in bad sequence contents (e.g., ODN 2010) had no stimulatory effecton NK activation. TABLE 10 ODN induction of NK Lytic Activity (LU) SEQ.ID. ODN Sequence (5′-3′) LU NO. Cells alone 0.01 — 1754 ACCATGGACGATCTGTTTCCCCTC 0.02 59 1758  TCTCCCAGCGTGCGCCAT 0.05 45 1761 TACCGCGTGCGACCCTCT 0.05 60 1776  ACCATGGACGAACTGTTTCCCCTC 0.03 61 1777 ACCATGGACGAGCTGTTTCCCCTC 0.05 62 1778  ACCATGGACGACCTGTTTCCCCTC 0.01 631779  ACCATGGACGTACTGTTTCCCCTC 0.02 64 1780  ACCATGGACGGTCTGTTTCCCCTC0.29 65 1781  ACCATGGACGTTCTGTTTCCCCTC 0.38 66 1823  GCATGACGTTGAGCT0.08 6 1824  CACGTTGAGGGGCAT 0.01 67 1825  CTGCTGAGACTGGAG 0.01 68 1828 TCAGCGTGCGCC 0.01 69 1829  ATGACGTTCCTGACGTT 0.42 70 1830² RANDOMSEQUENCE 0.25 1834  TCTCCCAGCGGGCGCAT 0.00 71 1836  TCTCCCAGCGCGCGCCAT0.46 72 1840  TCCATGTCGTTCCTGTCGTT 2.70 73 1841  TCCATAGCGTTCCTAGCGTT1.45 74 1842  TCGTCGCTGTCTCCGCTTCTT 0.06 75 1851  TCCTGACGTTCCTGACGTT2.32 76

[0132] TABLE 11 Induction of NK LU by Phosphorothioate CpG ODN with GoodMotifs SEQ. ODN¹ Sequence ID NO. expt. 1 expt. 2 expt. 3 Cells alone0.00 1.26 0.46 1840  TCCATGTCGTTCCTGTCGTT 73 2.33 ND ND 1960 TCCTGTCGTTCCTGTCGTT 77 ND 0.48 8.99 1961  TCCATGTCGTTTTTGTCGTT 78 4.031.23 5.08 1962  TCCTGTCGTTCCTTGTCGTT 52 ND 1.60 5.74 1963 TCCTTGTCGTTCCTGTCGTT 79 3.42 ND ND 1965  TCCTGTCGTTTTTTGTCGTT 53 0.460.42 3.48 1966  TCGTCGCTGTCTCCGCTTCTT 75 2.62 ND ND 1967 TCGTCGCTGTCTGCCCTTCTT 54 5.82 1.64 8.32 1968  TCGTCGCTGTTGTCGTTTCTT 553.77 5.26 6.12 1979² TCCATGTZGTTCCTGTZGTT 1.32 ND ND 1982 TCCAGGACTTCTCTCAGGTT 79 0.05 ND 0.98 1990  TCCATGCGTGCGTGCGTTTT 80 2.10ND ND 1991  TCCATGCGTTGCGTTGCGTT 81 0.89 ND ND 2002 TCCACGACGTTTTCGACGTT 82 4.02 1.31 9.79 2005  TCGTCGTTGTCGTTGTCGTT 47 ND4.22 12.75 2006  TCGTCGTTTTGTCGTTTTGTCGTT 56 ND 6.17 12.82 2007 TCGTCGTTGTCGTTTTGTCGTT 49 ND 2.68 9.66 2008  GCGTGCGTTGTCGTTGTCGTT 56 ND1.37 8.15 2010  GCGGCGGGCGGCGCGCGCCC 83 ND 0.01 0.05 2012 TGTCGTTTGTCGTTTGTCGTT 48 ND 2.02 11.61 2013  TGTCGTTGTCGTTGTCGTTGTCGTT84 ND 0.56 5.22 2014  TGTCGTTGTCGTTGTCGTT 60 ND 5.74 10.89 2015 TCGTCGTCGTCGTT 51 ND 4.53 10.13 2016  TGTCGTTGTCGTT 95 ND 6.54 8.06

[0133] Identification of Phosphorothioate ODN with Optimal CpG Motifsfor Activation of Human B Cell Proliferation.

[0134] The ability of a CpG ODN to induce B cell proliferation is a goodmeasure of its adjuvant potential. Indeed, ODN with strong adjuvanteffects generally also induce B cell proliferation. To determine whetherthe optimal CpG ODN for inducing B cell proliferation are the same asthose for inducing NK cell activity, similar panels of ODN (Table 12)were tested. The most consistent stimulation appeared with ODN 2006(Table 12). TABLE 12 Induction of Human B Cell Proliferation byPhosphorothioate CpG ODN Stimulation Index¹ DN Sequence (5′-3′) SEQ. ID.NO. expt. 1 expt. 2 expt. 3 expt. 4 expt. 5 1840 TCCATGTCGTTCCTGTCGTT 734 ND ND ND ND 1841 TCCATAGCGTTCCTAGCGTT 74 3 ND ND ND ND 1960TCCTGTCGTTCCTGTCGTT 77 ND 2.0 2.0 3.6 ND 1961 TCCATGTCGTTTTTGTCGTT 78 23.9 1.9 3.7 ND 1962 TCCTGTCGTTCCTTGTCGTT 52 ND 3.8 1.9 3.9 5.4 1963TCCTTGTCGTTCCTGTCGTT 79 3 ND ND ND ND 1965 TCCTGTCGTTTTTTGTCGTT 53 4 3.72.4 4.7 6.0 1967 TCGTCGCTGTCTGCCCTTCTT 54 ND 4.4 2.0 4.5 5.0 1968TCGTCGCTGTTGTCGTTTCTT 55 ND 4.0 2.0 4.9 8.7 1982 TCCAGGACTTCTCTCAGGTT 793 1.8 1.3 3.1 3.2 2002 TCCACGACGTTTTCGACGTT 86 ND 2.7 1.4 4.4 ND 2005TCGTCGTTGTCGTTGTCGTT 47 5 3.2 1.2 3.0 7.9 2006 TCGTCGTTTTGTCGTTTTGTCGTT46 4 4.5 2.2 5.8 8.3 2007 TCGTCGTTGTCGTTTTGTCGTT 49 3 4.0 4.2 4.1 ND2008 GCGTGCGTTGTCGTTGTCGTT 56 ND 3.0 2.4 1.6 ND 2010GCGGCGGGCGGCGCGCGCCC 83 ND 1.6 1.9 3.2 ND 2012 TGTCGTTTGTCGTTTGTCGTT 482 2.8 0 3.2 ND 2013 TGTCGTTGTCGTTGTCGTTGTCGTT 84 3 2.3 3.1 2.8 ND 2014TGTCGTTGTCGTTGTCGTT 50 3 2.5 4.0 3.2 6.7 2015 TCGTCGTCGTCGTT 51 5 1.82.6 4.5 9.4 2016 TGTCGTTGTCGTT 85 ND 1.1 1.7 2.7 7.3

[0135] Identification of Phosphorothioate ODN that Induce Human IL-12Secretion

[0136] The ability of a CpG ODN to induce IL-12 secretion is a goodmeasure of its adjuvant potential, especially in terms of its ability toinduce a Th1 immune response, which is highly dependent on IL-12.Therefore, the ability of a panel of phosphorothioate ODN to induceOIL-12 secretion from human PBMC in vitro (Table 13) was examined. Theseexperiments showed that in some human PBMC, most CpG ODN could induceIL-12 secretion (e.g., expt. 1). However, other donors responded to justa few CpG ODN (E.g., expt. 2). ODN 2006 was a consistent inducer of IL12secretion from most subjects (Table 13). TABLE 13 Induction of HumanIL-2 Secretion by Phosphorothioate CpG ODN IL-12 (pg/ml) SEQ ID expt.expt. ODN1 Sequence (5′-3′) NO. 1 2 Cells 0 0 alone 1962TCCTGTCGTTCCTTGTCGTT 52 19 0 1965 TCCTGTCGTTTTTTGTCGTT 53 36 0 1967TCGTCGCTGTCTGCCCTTCTT 54 41 0 1968 TCGTCGCTGTTGTCGTTTCTT 55 24 0 2005TCGTCGTTGTCGTTGTCGTT 47 25 0 2006 TCGTCGTTTTGTCGTTTTGTCGTT 46 29 15 2014TGTCGTTGTCGTTGTCGTT 50 28 0 2015 TCGTCGTCGTCGTT 51 14 0 2016TGTCGTTGTCGTT 85 3 0

[0137] Identification of B cell and Monocyte/NK Cell-SpecificOligonucleotides

[0138] As shown in FIG. 6, CpG DNA can directly activate highly purifiedB cells and monocytic cells. There are many similarities in themechanism through which CpG DNA activates these cell types. For example,both require NFkB activation as explained further below.

[0139] In further studies of different immune effects of CpG DNA, it wasfound that there is more than one type of CpG motif. Specifically, olio1668, with the best mouse B cell motif, is a strong inducer of both Bcell and natural killer (NK) cell activation, while olio 1758 is a weakB cell activator, but still induces excellent NK responses (Table 14).TABLE 14 Different CpG Motifs Stimulate Optimal Murine B Cell and NKActivation B Cell NK ODN¹ Sequence Activation Activation² 1668TCCATGACGTTCCTGATGCT 42,849 2.52 (SEQ.ID.NO:7) 1758 TCTCCCAGCGTGCGCCAT1,747 6.66 (SEQ.ID.NO.45) NONE 367 0.00

[0140] Teleological Basis of Immunostimulatory Nucleic Acids

[0141] Vertebrate DNA is highly methylated and CpG dinucleotides areunder represented. However, the stimulatory CpG motif is common inmicrobial genomic DNA, but quite rare in vertebrate DNA. In addition,bacterial DNA has been reported to induce B cell proliferation andimmunoglobulin (Ig) production, while mammalian DNA does not (Messina,J. P. et al., J. Immunol. 147:1759 (1991)). Experiments furtherdescribed in Example 3, in which methylation of bacterial DNA with CpGmethylase was found to abolish mitogenicity, demonstrates that thedifference in CpG status is the cause of B cell stimulation by bacterialDNA. This data supports the following conclusion: that unmethylated CpGdinucleotides present within bacterial DNA are responsible for thestimulatory effects of bacterial DNA.

[0142] Teleologically, it appears likely that lymphocyte activation bythe CpG motif represents an immune defense mechanism that can therebydistinguish bacterial from host DNA. Host DNA, which would commonly bepresent in many anatomic regions and areas of inflammation due toapoptosis (cell death), would generally induce little or mo lymphocyteactivation due to CpG suppression and methylation. However, the presenceof bacterial DNA containing unmethylated CpG motifs can cause lymphocyteactivation precisely in infected anatomic regions, where it isbeneficial. This novel activation pathway provides a rapid alternativeto T cell dependent antigen specific B cell activation. Since the CpGpathway synergizes with B cell activation through the antigen receptor,B cells bearing antigen receptor specific for bacterial antigens wouldreceive on e activation signal through cell membrane Ig and a secondsignal from bacterial DNA, and would therefore tend to be preferentiallyactivated. The interrelationship of this pathway with other pathways ofB cell activation provide a physiologic mechanism employing a polyclonalantigen to induce antigen-specific responses.

[0143] However, it is likely that B cell activation would not be totallynonspecific. B cells bearing antigen receptors specific for bacterialproducts could receive one activation signal through cell membrane Ig,and a second from bacterial DNA, thereby more vigorously triggeringantigen specific immune responses. As with other immune defensemechanisms, the response to bacterial DNA could have undesirableconsequences in some settings. For example, autoimmune responses to selfantigens would also tend to be preferentially triggered by bacterialinfections, since autoantigens could also provide a second activationsignal to autoreactive B cells triggered by bacterial DNA. Indeed theinduction of autoimmunity by bacterial infections is a common clinicalobservance. For example, the autoimmune disease systemic lupuserythematosus, which is: i) characterized by the production of anti-DNAantibodies; ii) induced by drugs which inhibit DNA methyltransferase(Cornaccia, E. J. et al., J. Clin. Invest. 92:38 (1993)); and iii)associated with reduced DNA methylation (Richardson, B., L. et al.,Arth. Rheum 35:647 (1992)), is likely triggered at least in part byactivation of DNA-specific B cells through stimulatory signals providedby CpG motifs, as well as by binding of bacterial DNA to antigenreceptors.

[0144] Further, sepsis, which is characterized by high morbidity andmortality due to massive and nonspecific activation of the immune systemmay be initiated by bacterial DNA and other products released from dyingbacteria that reach concentrations sufficient to directly activate manylymphocytes. Further evidence of the role of CpG DNA in the sepsissyndrome is described in Cowdery, J., et. al., (1996) the Journal ofImmunology 156:4570-4575.

[0145] Unlike antigens that trigger B cells through their surface Igreceptor, CpG-ODN did not induce any detectable Ca²⁺ flux, changes inprotein tyrosine phosphorylation, or IP 3 generation. Flow cytometrywith FITC-conjugated ODN with or without a CpG motif was performed asdescribed in Zhao, Q et al., (Antisense Research and Development 3:53-66(1993)), and showed equivalent membrane binding, cellular uptake,efflux, and intracellular localization. This suggests that there may notbe cell membrane proteins specific for CpG ODN. Rather than actingthrough the cell membrane, that data suggests that unmethylated CpGcontaining oligonucleotides require cell uptake for activity: ODNcovalently linked to a solid Teflon support were nonstimulatory, as werebiotinylated ODN immobilized on either avidin beads or avidin coatedpetri dishes. CpG ODN conjugated to either FITC or biotin retained fullmitogenic properties, indicated no stearic hindrance.

[0146] Recent data indicate the involvement of the transcription factorNFkB as a direct or indirect mediator of the CpG effect. For example,within 15 minutes of treating B cells or monocytes with CpG DNA, thelevel of NFkB binding activity is increased (FIG. 7). However, it is notincreased by DNA that does not contain CpG motifs. In addition, it wasfound that two different inhibitors of NFkB activation, PDTC andgliotoxin, completely block the lymphocyte stimulation by CpG DNA asmeasured by B cell proliferation or monocytic cell cytokine secretion,suggesting that NFkB activation is required for both cell types.

[0147] There are several possible mechanisms through which NFkB can beactivated. These include through activation of various protein kinases,or through the generation of reactive oxygen species. No evidence forprotein kinase activation induced immediately after CpG DNA treatment ofB cells or monocytic cells have been found, and inhibitors of proteinkinase A, protein kinase C, and protein tyrosine kinases had no effectson the CpG induced activation. k However, CpG DNA causes a rapidinduction of the production of reactive oxygen species in both B cellsand monocytic cells, as detected by the sensitive fluorescent dyedihydrorhodamine 123 as described in Royall, J. A., and Ischiropoulos,H. (Archives of Biochemistry and Biophysics 302:348-355 (1993)).Moreover, inhibitors of the generation of these reactive oxygen speciescompletely block the induction of NFkB and the later induction of cellproliferation and cytokine secretion by CpG DNA.

[0148] Work backwards, the next question was how CpG DNA leads to thegeneration of reactive oxygen species so quickly. Previous studies bythe inventors demonstrated that oligonucleotides and plasmid orbacterial DNA are taken up by cells into endosomes. These endosomesrapidly become acidified inside the cell. To determine whether thisacidification step may be important in the mechanism through which CpGDNA activates reactive oxygen species, the acidification step wasblocked with specific inhibitors of endosome acidification includingchloroquine, monensin, and bafilomycin, which work through differentmechanisms. FIG. 8A shows the results from a flow cytometry study usingmouse B cells with the dihydrorhodamine 123 dye to determine levels ofreactive oxygen species. The dye only sample in Panel A of the figureshows the background level of cells positive for the dye at 28.6%. asexpected, this level of reactive oxygen species was greatly increased to80% in the cells treated for 20 minutes with PMA and ionomycin, apositive control (Panel B). The cells treated with the CpG oligo alsoshowed an increase in the level of reactive oxygen species such thatmore than 50% of the cells became positive (Panel D). However, cellstreated with an oligonucleotide with the identical sequence except thatthe CpG was switched did not show this significant increase in the levelof reactive oxygen species (Panel E).

[0149] In the presence of chloroquine, the results are very different(FIG. 8B). Chloroquine slightly lowers the background level of reactiveoxygen species in the cells such that the untreated cells in Panel Ahave only 4.3% that are positive. Chloroquine completely abolishes theinduction of reactive oxygen species in the cells treated with CpG DNA(Panel B) but does not reduce the level of reactive oxygen species inthe cells treated with PMA and ionomycin (Panel E). This demonstratesthat unlike the PMA plus ionomycin, the generation of reactive oxygenspecies following treatment of B cells with CpG DNA requires that theDNA undergo an acidification step in the endosomes. This is a completelynovel mechanism of leukocyte activation. Chloroquine, monensin, andbafilomycin also appear to block the activation of NFkB by CpG DNA aswell as the subsequent proliferation and induction of cytokinesecretion.

[0150] Chronic Immune Activation by CpG DNA and Autoimmune Disorders

[0151] B cell activation by CpG DNA synergizes with signals through theB cell receptor. This raises the possibility that DNA-specific B cellsmay be activated by the concurrent binding of bacterial DNA to theirantigen receptor, and by the co-stimulatory CpG-mediated signals. Inaddition, CpG DNA induces B cells to become resistant to apoptosis, amechanism thought to be important for preventing immune responses toself antigens, such as DNA. Indeed, exposure to bDNA can triggeranti-DNA Ab production. Given this potential ability of CpG DNA topromote autoimmunity, it is therefore noteworthy that patients with theautoimmune disease systemic lupus erythematosus have persistentlyelevated levels of circulating plasma DNA which is enriched inhypomethylated CpGs. These findings suggest a possible role for chronicimmune activation by CpG DNA in lupus etiopathogenesis.

[0152] A class of medications effective in the treatment of lupus isantimalarial drugs, such as chloroquine. While the therapeutic mechanismof these drugs has been unclear, they are known to inhibit endosomalacidification. Leukocyte activation by CpG DNA is not medicated throughbinding to a cell surface receptor, but requires cell uptake, whichoccurs via adsorptive endocytosis into an acidifiedchloroquine-sensitive intracellular compartment. This suggested thehypothesis that leukocyte activation by CpG DNA may occur in associationwith acidified endosomes, and might even be pH dependent. To test thishypothesis specific inhibitors of DNA acidification were applied todetermine whether B cells or monocytes could respond to CpG DNA ifendosomal acidification was prevented.

[0153] The earliest leukocyte activation event that was detected inresponse to CpG DNA is the production of reactive oxygen species (ROS),which is induced within five minutes in primary spleen cells and both Band monocyte cell lines. Inhibitors of endosomal acidification includingchloroquine, bafilomycin A, and monensin, which have differentmechanisms of action, blocked the CpG-induced generation of ROS, but hadno effect on ROS generation mediated by PMA, or ligation of CD40 or IgM.These studies show that ROS generation is a common event in leukocyteactivation through diverse pathways. This ROS generation is generallyindependent of endosomal acidification, which is required only for theROS response to CpG DNA. ROS generation in response to CpG is notinhibited by the NFκB inhibitor gliotoxin, confirming that it is notsecondary to NFκB activation.

[0154] To determine whether endosomal acidification of CpG DNA was alsorequired for its other immune stimulatory effects were performed. BothLPS and CpG DNA induce similar rapid NFκB activation, increases inproto-oncogene mRNA levels, and cytokine secretion. Activation of NFκBby DNA depended on CpG motifs since it was not induced by bDNA treatedwith CpG methylase, nor by ODN in which bases were switched to disruptthe CpGs. Supershift experiments using specific antibodies indicatedthat the activated NFκB complexes included the p50 and p65 components.Not unexpectedly, NFκB activation in LPS- or CpG-treated cells wasaccompanied by the degradation of IκBα and IκBβ. However, inhibitors ofendosomal acidification selectively blocked all of the CpG-induced butnone of the LPS-induced cellular activation events. The very lowconcentration of chloroquine (<10 μM) that has been determined toinhibit CpG-mediated leukocyte activation is noteworthy since it is wellbelow that required for antimalarial activity and other reported immuneeffects (e.g., 100-1000 μM). These experiments support the role of apH-dependent signaling mechanism in mediating the stimulatory effects ofCpG DNA. TABLE 15 Specific blockade of CpG-induced TNF-α and IL-12expression by inhibitors of endosomal acidification or NFκB activationInhibitors: Bafilomycin Chloroquine Monensin NAC TPCK GliotoxinBisgliotoxin Medium (250 nM) (2.5 μg/ml) (10 μM) (50 mM) (50 μM) (0.1μg/ml) (0.1 μg/ml) activators TNF-α IL-12 TNF-α IL-12 TNF-α IL-12 TNF-αIL-12 TNF-α TNF-α TNF-α TNF-α Medium 37 147 46 102 27 20 22 73 10 24 1741 CpG 455 17,114 71 116 28 6 49 777 54 23 31 441 ODN LPS 901 22,4851370 4051 1025 12418 491 4796 417 46 178 1120 #NO: 10) at 2 μM or LPS(10 μg/ml) for 4 hr (TNF-α) or 24 hr (IL-12) at which time thesupernatant was harvested. ELISA for IL-12 or TNF-α (pg/ml) wasperformed on the supernatants essentially as described (A. K. Krieg, A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G.Koretzky and D. Klinman, Nature #374, 546 (1995); Yi, A. -K., D. M.Klinman, T. L. Martin, S. Matson and A. M. Krieg, J. Immunol., 157,5394-5402 (1996); Krieg, A. M., J. Lab. Clin. Med., 128, 128-133 (1996).Cells cultured with ODN that lacked CpG motifs did not induce cytokinesecretion. Similar specific inhibition of CpG responses was seen withIL-6 assays, #and in experiments using primary spleen cells or the Bcell lines CH 12.LX and WEHI-231.2.5 μg/ml of chloroquine is equivalentto <5 μM. Other inhibitors of NF-κB activation including PDTC andcalpain inhibitors I and II gave similar results to the inhibitorsshown. The results shown are representative of those obtained in tendifferent experiments.

[0155] Excessive immune activation by CpG motifs may contribute to thepathogenesis of the autoimmune disease systemic lupus erythematosus,which is associated with elevated levels of circulating hypomethylatedCpG DNA. Chloroquine and related antimalarial compounds are effectivetherapeutic agents for the treatment of systemic lupus erythematosus andsome other autoimmune diseases, although their mechanism of action hasbeen obscure. Our demonstration of the ability of extremely lowconcentrations of chloroquine to specifically inhibit CpG-mediatedleukocyte activation suggests a possible new mechanism for itsbeneficial effect. It is noteworthy that lupus recurrences frequentlyare thought to be triggered by microbial infection. Levels of bDNApresent in infected tissues can be sufficient to induce a localinflammatory response. Together with the likely role of CpG DNA as amediator of the sepsis syndrome and other diseases our studies suggestpossible new therapeutic applications for the antimalarial drugs thatact as inhibitors of endosomal acidification.

[0156] CpG-induced ROS generation could be an incidental consequence ofcell activation, or a signal that mediates this activation. The ROSscavenger N-acetyl-L-cysteine (NAC) blocks CpG-induced NFκB activation,cytokine production, and B cell proliferation, suggesting a casual rolefor ROS generation in these pathways. These data are compatible withprevious evidence supporting a role for ROS in the activation of NFκB.WEHI-231 B cells (5×10⁵ cells/ml) were precultured for 30 minutes withor without chloroquine (5 μg/ml [<10 μM]) or gliotoxin (0.2 μg/ml). Cellaliquots were then cultured as above for 10 minutes in RPMI medium withor without a CpG ODN (1826) or non-CpG ODN (1911) at 1 μM or phorbolmyristate acetate (PMA) plus ionomycin (iono). Cells were then stainedwith dihydrorhodamine-123 and analyzed for intracellular ROS productionby flow cytometry as described (A. K. Krieg, A.-K. Yi, S. Matson, T. J.Waldschmidt, G. A. Bishop, R. Teasdale, G. Koretzky and D. Klinman,Nature 374, 546 (1995); Yi, A.-K., D. M. Klinman, T. L. Martin, S.Matson and A. M. Krieg, J. Immunol., 157, 5394-5402 (1996); Krieg, A. M,J. Lab. Clin. Med., 128, 128-133 (1996)). J 1774 cells, a monocyticline, showed similar pH-dependent CpG induced ROS responses. Incontrast, CpG DNA did not induce the generation of extracellular ROS,nor any detectable neutrophil ROS. The concentrations of chloroquine(and those used with the other inhibitors of endosomal acidification)prevented acidification of the internalized CpG DNA using fluoresceinconjugated ODN as described by Tonkinson, et al., (Nucl. Acids Res. 22,4268 (1994); A. M. Krieg, In: Delivery Strategies for AntisenseOligonucleotide Therapeutics. Editor, S. Akhtar, CRC Press, Inc., pp.177(1995)). At higher concentrations than those required to inhibitendosomal acidification, nonspecific inhibitory effects were observed.Each experiment was performed at least three times with similar results.

[0157] While NFκB is known to be an important regulator of geneexpression, it's role in the transcriptional response to CpG DNA wasuncertain. To determine whether this NFκB activation was required forthe CpG mediated induction of gene expression cells were activated withCpG DNA in the presence or absence of pyrrolidine dithiocarbamate(PDTC), an inhibitor of IκB phosphorylation. These inhibitors of NFκBactivation completely blocked the CpG-induced expression ofprotooncogene and cytokine mRNA and protein, demonstrating the essentialrole of NFκB as a mediator of these events. None of the inhibitorsreduced cell viability under the experimental conditions used in thesestudies. A J774, a murine monocyte cell line, was cultured in thepresence of calf thymus (CT), E. Coli (EC), or methylated E. Coli (mEC)DNA (methylated with CpG methylase as^(described)) at 5 μg/ml or a CpGoligodeoxynucleotide (ODN 1826; Table 15) or a non-CpG ODN (ODN 1745;TCCATGAGCTTCCTGAGTCT, SEQ. ID. NO: 8) at 0.75 μM for 1 hr, followingwhich the cells were lysed and nuclear extracts prepared. A doublestranded ODN containing a consensus NFκB site was 5′ radiolabeled andused as a probe for EMSA essentially as described (J. D. Dignam, R. M.Lebovitz and R. G. Roeder, Nucleic Acids Res. 11, 1475 (1983); M.Briskin, M. Damore, R. Law, G. Lee, P. W. Kincade, C. H. Sibley, M.Kuehl and R. Wall, Mol. Cell. Biol. 10, 422 (1990)). The position of thep50/p65 heterodimer was determined by supershifting with specific Ab top65 and p50 (Santa Cruz Biotechnology, Santa Cruz, Calif.). Chloroquineinhibition of CpG-induced but not LPS-induced NFκB activation wasestablished using J774 cells. The cells were precultured for 2 hr in thepresence or absence of chloroquine (20 μg/ml) and then stimulated asabove for 1 hr with either EC DNA, CpG ODN, non-CpG ODN or LPS (1μg/ml). Similar chloroquine sensitive CpG-induced activation of NFκB wasseen in a B cell line, WEHI-231 and primary spleen cells. Theseexperiments were performed three times over a range of chloroquineconcentrations form 2.5 to 20 μg/ml with similar results.

[0158] It was also established that CpG-stimulated mRNA expressionrequires endosomal acidification and NFκB activation in B cells andmonocytes. J774 cells (2×10⁶ cells/ml) were cultured for 2 hr in thepresence or absence of chloroquine (2.5 μg/ml [<5 μM]) orN-tosyl-L-phenylalanine chlorometryl ketone (TPCK: 50 μM), aserine/threonine protease inhibitor that prevents IκB proteolysis andthus blocks NFκB activation. Cells were then stimulated with theaddition of E. Coli DNA (EC: 50 μg/ml), calf thymus DNA (CT: 50 μg/ml),LPS (10 μg/ml), CpG ODN (1826; 1 μM), or control non CpG ODN (1911; 1μM) for 3 hr. WEHI-231 B cells (5×10⁵ cells/ml) were cultured in thepresence or absence of gliotoxin (0.1 μg/ml) or bisgliotoxin (0.1 μg/ml)for 2 hrs and then stimulated with a CpG ODN (1826), or control non-CpGODN (1911; TCCAGGACTTTCCTCAGGTT, SEQ. ID. NO.97) at 0.5 μM for 8 hr. Inboth cases, cells were harvested and RNA was prepared using RNAzolfollowing the manufacturer's protocol. Multi-probe RNASE protectionassay was performed as described (A.-K. Yi, P. Hornbeck, D. E. Lafrenzand A. M. Krieg, J Immunol., 157, 4918-4925 (1996). Comparable amountsof RNA were loaded into each lane by using ribosomal mRNA as a loadingcontrol (L32). These experiments were performed three times with similarresults.

[0159] The results indicate that leukocytes respond to CpG DNA through anovel pathway involving the pH-dependent generation of intracellularROS. The pH dependent step may be the transport or processing of the CpGDNA, the ROS generation, or some other event. ROS are widely thought tobe second messengers in signaling pathways in diverse cell types, buthave not previously been shown to mediate a stimulatory signal in Bcells.

[0160] Presumably, there is a protein in or near the endosomes thatspecifically recognizes DNA containing CpG motifs and leads to thegeneration of reactive oxygen species. To detect any protein in the cellcytoplasm that may specifically bind CpG DNA, electrophoretic mobilityshift assays (EMSA) were used with 5′ radioactively labeledoligonucleotides with or without CpG motifs. A band was found thatappears to represent a protein binding specifically to single strandedoligonucleotides that have CpG motifs, but not to oligonucleotides thatlack CpG motifs or to oligonucleotides in which the CpG motif has beenmethylated. This binding activity is blocked if excess ofoligonucleotides that contain the NFκB binding site was added. Thissuggests that an NFκB or related protein is a component of a protein orprotein complex that binds the stimulatory CpG oligonucleotides.

[0161] No activation of CREB/ATF proteins was found at time points whereNFκB was strongly activated. These data therefore do not provide proofthe NFκB proteins actually bind to the CpG nucleic acids, but ratherthat the proteins are required in some way for the CpG activity. It ispossible that a CREB/ATF or related protein may interact in some waywith NFκB proteins or other proteins thus explaining the remarkablesimilarity in the binding motifs for CREB proteins and the optimal CpGmotif. It remains possible that the oligos bind to a CREB/ATF or relatedprotein, and that this leads to NFκB activation.

[0162] Alternatively, it is very possible that the CpG nucleic acids maybind to one of the TRAF proteins that bind to the cytoplasmic region ofCD40 and mediate NFκB activation when CD40 is cross-linked. Examples ofsuch TRAF proteins include TRAF-2 and TRAF-5.

[0163] Method for Making Immunostimulatory Nucleic Acids

[0164] For use in the instant invention, nucleic acids can besynthesized de novo using any of a number of procedures well known inthe art. For example, the b-cyanoethyl phosphoramidite method (S. L.Beaucage and M. H. Caruthers, (1981) Tet. Let. 22:1859); nucleosideH-phosphonate method (Garegg et al., (1986) Tet. Let. 27:4051-4054;Froehler et al., (1986) Nucl. Acid. Res 14:5399-5407; Garegg eg al.,(1986) Tet. Let. 27:4055-4058, Gaffney et al., (1988) Tet. Let.29:2619-2622). These chemistries can be performed by a variety ofautomated oligonucleotide synthesizers available in the market.Alternatively, oligonucleotides can be prepared from existing nucleicacid sequences (e.g. genomic or cDNA) using known techniques, such asthose employing restriction enzymes, exonucleases or endonucleases.

[0165] For use in vivo, nucleic acids are preferably relativelyresistant to degradation (e.g. via endo- and exo-nucleases). Secondarystructures, such as stem loops, can stabilize nucleic acids againstdegradation. Alternatively, nucleic acid stabilization can beaccomplished via phosphate backbone modifications. A preferredstabilized nucleic acid has at least a partial phosphorothioate modifiedbackbone. Phosphorothioates may be synthesized using automatedtechniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made e.g. as describedin U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which thecharged oxygen moiety is alkylated as described in U.S. Pat. No.5,023,243 and European Patent No. 092,574) can be prepared by automatedsolid phase synthesis using commercially available reagents. Methods formaking other DNA backbone modifications and substitutions have beendescribed (Uhlmann, E. And Peyman, A. (1990) Chem. Rev. 90:544;Goodchild, J. (I 990) Bioconjugate Chem. 1: 165). 2′-O-methyl nucleicacids with CpG motifs also cause immune activation, as doethoxy-modified CpG nucleic acids. In fact, no backbone modificationshave been found that completely abolish the CpG effect, although it isgreatly reduced by replacing the C with a 5-methyl C.

[0166] For administration in vivo, nucleic acids may be associated witha molecule that results in higher affinity binding to target cell (e.g.B-cell, monocytic cell and natural killer (NK) cell) surfaces and/orincreased cellular uptake by target cells to form a “nucleic aciddelivery complex”. Nucleic acids can be ionically, or covalentlyassociated with appropriate molecules using techniques which are wellknown in the art. A variety of coupling or crosslinking agents can besued e.g. Protein A, carbodiimide, andN-succinimidyl-3-(2-pyridyidithio) propionate (SPDP). Nucleic acids canalternatively be encapsulated in liposomes or virosomes using well-knowntechniques.

[0167] Therapeutic Uses of Immunostimulatory Nucleic Acid Molecules

[0168] Based on their immunostimulatory properties, nucleic acidmolecules containing at least one unmethylated CpG dinucleotide can beadministered to a subject in vivo to treat an “immune systemdeficiency”. Alternatively, nucleic acid molecules containing at leastone unmethylated CpG dinucleotide can be contacted with lymphocytes(e.g. B cells, monocytic cells or NK cells) obtained from a subjecthaving an immune system deficiency ex vivo and activated lymphocytes canthen be re-implanted in the subject.

[0169] As reported herein, in response to unmethylated CpG containingnucleic acid molecules, an increased number of spleen cells secreteIL-6, IL-12, IFNγ, IFN-α, IFN-β, IL-1, IL-3, IL-10, TNF-α, TNF-β,GM-CSF, RANTES, and probably others. The increased IL-6 expression wasfound to occur in B cells, CD4⁺T cells and monocytic cells.

[0170] Immunostimulatory nucleic acid molecules can also be administeredto a subject in conjunction with a vaccine to boost a subject's immunesystem and thereby effect a better response from the vaccine. Preferablythe immunostimulatory nucleic acid molecule is administered slightlybefore or at the same time as the vaccine. A conventional adjuvant mayoptionally be administered in conjunction with the vaccine, which isminimally comprised of an antigen, as the conventional adjuvant mayfurther improve the vaccination by enhancing antigen absorption.

[0171] When the vaccine is a DNA vaccine at least two componentsdetermine its efficacy. First, the antigen encoded by the vaccinedetermines the specificity of the immune response. Second, if thebackbone of the plasmid contains CpG motifs, its functions as anadjuvant for the vaccine. Thus, CpG DNA acts as an effective “dangersignal” and causes the immune system to respond vigorously to newantigens in the area. This mode of action presumably results primarilyfrom the stimulatory local effects of CpG DNA on dendritic cells andother “professional” antigen presenting cells, as well as from theco-stimulatory effects on B cells.

[0172] Immunostimulatory oligonucleotides and unmethylated CpGcontaining vaccines, which directly activate lymphocytes andco-stimulate an antigen-specific response, are fundamentally differentfrom conventional adjuvants (e.g. aluminum precipates), which are inertwhen injected alone and are thought to work through absorbing theantigen and thereby presenting it more effectively to immune cells.Further conventional adjuvants only work for certain antigens, onlyinduce an antibody (humoral) immune response (Th2), and are very poor atinducing cellular immune responses (Th1). For many pathogens, thehumoral response contributes little to protection, and can even bedetrimental.

[0173] In addition, an immunostimulatory oligonucleotide can beadministered prior to along with or after administration of achemotherapy or immunotherapy to increase the responsiveness of themalignant cells to subsequent chemotherapy or immunotherapy or to speedthe recovery of the bone marrow through induction of restorativecytokines such as GM-CSF. CpG nucleic acids also increase natural killercell lytic activity and antibody dependent cellular cytotoxicity (ADCC).Induction of NK activity and ADCC may likewise be beneficial in cancerimmunotherapy, alone or in conjunction with other treatments.

[0174] Another use of the described immunostimulatory nucleic acidmolecules is in desensitization therapy for allergies, which aregenerally caused by IgE antibody generation against harmless allergens.The cytokines that are induced by unmethylated CpG nucleic acids arepredominantly of a class called “Th1” which is most marked; by acellular immune response and is associated with Il-12 and IFN-γ. Theother major type of immune response is termed a Th2 immune response,which is associated with more of an antibody immune response and withthe production of IL-4, Il-5 and IL-10. In general, it appears thatallergic diseases are mediated by Th2 type immune responses andautoimmune diseases by Th1 immune response. Based on the ability of theimmunostimulatory nucleic acid molecules to shift the immune response ina subject from a Th2 (which is associated with production of IgEantibodies and allergy) to a Th1 response (which is protective againstallergic reactions), an effective dose of an immunostimulatory nucleicacid (or a vector containing a nucleic acid) alone or in conjunctionwith an allergen can be administered to a subject to treat or prevent anallergy.

[0175] Nucleic acids containing unmethylated CpG motifs may also havesignificant therapeutic utility in the treatment of asthma. Th2cytokines, especially IL-4 and IL-5 are elevated in the airways ofasthmatic subjects. These cytokines promote important aspects of theasthmatic inflammatory response, including IgE isotype switching,eosinophil chemotaxis and activation and mast cell growth. Th1cytokines, especially IFN-γ and IL-12, can suppress the formation of Th2clones and production of Th2 cytokines.

[0176] As described in detail in the following Example 12,oligonucleotides containing an unmethylated CpG motif (I, e,.TCCATGACGTTCCTGACGTT; SEQ ID NO. 10) but not a control oligonucleotide(TCCATGAGCTTCCTGAGTCT; SEQ ID NO. 8) prevented the development of aninflammatory cellular infiltrate and eosinophilia in a murine model ofasthma. Furthermore, the suppression of eosinophilic inflammation wasassociated with a suppression of a Th2 response and induction of a Th1response.

[0177] For use in therapy, an effective amount of an appropriateimmunostimulatory nucleic acid molecule alone or formulated as adelivery complex can be administered to a subject by any mode allowingthe oligonucleotide to be taken up by the appropriate target cells(e.g., B-cells and monocytic cells). Preferred routes of administrationinclude oral and transdermal (e.g., via a patch). Examples of otherroutes of administration include injection (subcutaneous, intravenous,parenteral, intraperitoneal, intrathecal, etc.). The injection can be ina bolus or a continuous infusion.

[0178] A nucleic acid alone or as a nucleic acid delivery complex can beadministered in conjunction with a pharmaceutically acceptable carrier.As used herein, the phrase “pharmaceutically acceptable carrier” isintended to include substances that can be coadministered with a nucleicacid or a nucleic acid delivery complex and allows the nucleic acid toperform its indicated function. Examples of such carriers includesolutions, solvents, dispersion media, delay agents, emulsions and thelike. The use of such media for pharmaceutically active substances arewell known in the art. Any other conventional carrier suitable for usewith the nucleic acids falls within the scope of the instant invention.

[0179] The term “effective amount” of a nucleic acid molecule refers tothe amount necessary or sufficient to realize a desired biologic effect.For example, an effective amount of a nucleic acid containing at leastone unmethylated CpG for treating an immune system deficiency could bethat amount necessary to eliminate a tumor, cancer, or bacterial, viralor fungal infection. An effective amount for use as a vaccine adjuvantcould be the amount useful for boosting a subjects immune response to avaccine. An “effective amount” for treating asthma can be that amount;useful for redirecting a Th2 type of immune response that is associatedwith asthma to a Th1 type of response. The effective amount for anyparticular application can vary depending on such factors as the diseaseor condition being treated, the particular nucleic acid beingadministered (e.g. the number of unmethylated CpG motifs or theirlocation in the nucleic acid), the size of the subject, or the severityof the disease or condition. One of ordinary skill in the art canempirically determine the effective amount of a particularoligonucleotide without necessitating undue experimentation.

[0180] The present invention is further illustrated by the followingExamples, which in no way should be construed as further limiting. Theentire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

EXAMPLES Example 1 Effects of ODNs on B Cell Total RNA Synthesis andCell Cycle

[0181] B cells were purified from spleens obtained from 6-12 week oldspecific pathogen free DBA/2 or BXSB mice (bred in the University ofIowa animal care facility; no substantial strain differences were noted)that were depleted of T cells with anti-Thy-1.2 and complement andcentrifugation over lymphocyte M(Cedarlane Laboratories, Hornby,Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4⁺ orCD8⁺ cells. 8×10⁴B cells were dispensed in triplicate into 96 wellmicrotiter plates in 100 μl RPMI containing 10% FBS (heat inactivated to65° C. for 30 min.), 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100ug/ml streptomycin, and 2 mM L-glutamate. 20 μM ODN were added at thestart of culture for 20 h at 37° C., cells pulsed with 1 μCi of ³Huridine, and harvested and counted 4 hr later. Ig secreting B cells wereenumerated using the ALISA spot assay after culture of whole spleencells with ODN at 20 μM for 48 hr. Data, reported in Table 1, representsthe stimulation index compared to cell cultured without ODN. ³Hthymidine incorporation assays showed similar results, but with somenonspecific inhibition by thymidine released from degraded ODN (Matson.S and A. M. Krieg (1992) Nonspecific suppression of ³H-thymidineincorporation by control oligonucleotides. Antisense Research andDevelopment 2:325).

Example 2 Effects of ODN on Production of IgM from B Cells

[0182] Single cell suspensions form the spleens of freshly killed micewere treated with anti-Thyl, anti-CD4, and anti-CD8 and complement bythe method of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting Bcells (<02% T cell contamination) were isolated from the 63-70% band ofa discontinuous Percoll gradient by the procedure of DeFranco et al, J.Exp. Med. 155:1523 (1982). These were cultured as described above in 30μg/ml LPS for 48 hr. The number of B cells actively secreting IgM wasmaximal at this time point, as determined by ELIspot assay (Klinman, D.M. et al. J. Immunol 144.506 (1990)). In that assay, B cells wereincubated for 6 hrs on anti-Ig coated microtiter plates. The Ig theyproduced (>99% IgM) was detected using phosphatase-labeled anti-Ig(Southern Biotechnology Associated, Birmingham, Ala.). The antibodiesproduced by individual B cells were visualized by addition of BCIP(Sigma Chemical Co., St. Louis Mo.) which forms an insoluble blueprecipitate in the presence of phosphatase. The dilution of cellsproducing 20-40 spots/well was used to determine the total number ofantibody-secreting B cells/sample. All assays were performed intriplicate (data reported in Table 1). In some experiments, culturesupernatants were assayed for IgM by ELISA, and showed similar increasedin response to CpG-ODN.

Example 3 B cell Stimulation by Bacterial DNA

[0183] DBA/2 B cells were cultured with no DNA or 50 μg/ml ofa(Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NSF/N mousespleen genomic DNAs for 48 hours, then pulsed with ³H thymidine for 4hours prior to cell harvest. Duplicate DNA samples were digested withDNASE I for 30 minutes at 37° C. prior to addition to cell cultures. Ecoli DNA also induced an 8.8 fold increase in the number of IgMsecreting B cells by 48 hours using the ELISAspot assay.

[0184] DBA/2 B cells were cultured with either no additive, 50 μg/ml LPSor the ODN 1; 1a; 4; or 4a at 20 μM. Cells were cultured and harvestedat 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with5, 10, 20, 40 or 80 μM of ODN 1; 1a; 4; or 4a or LPS. In thisexperiment, wells with no ODN had 3833 cpm. Each experiment wasperformed at least three times with similar results. Standard deviationsof the triplicate wells were <5%.

Example 4 Effects of ODN on Natural Killer (NK) Activity

[0185] 10×10⁶ C57BL/6 spleen cells were cultured in two ml RPMI(supplemented as described for Example 1) with or without 40 μM CpG ornon-CpG ODN for forty-eight hours. Cells were washed, and then used aseffector cells in a short term ⁵¹Cr release assay with YAC-1 and 2C11,two NK sensitive target cell lines (Ballas, Z. K. et al. (1993) j.IMMUNOL. 150:17). Effector cells were added at various concentrations to10⁴ ⁵¹Cr-labeled target cells in V-bottom microtiter plates in 0.2 ml,and incubated in 5% CO₂ for 4 hr. At 37° C. Plates were thencentrifuged, and an aliquot of the supernatant counted forradioactivity. Percent specific lysis was determined by calculating theratio of the ⁵¹Cr released in the presence of effector cells minus the⁵¹Cr released when the target cells are cultured alone, over the totalcounts released after cell lysis in 2% acetic acid minus the ⁵¹Cr cpmreleased when the cells are cultured alone.

Example 5 In vivo Studies with CpG Phosphorothioate ODN

[0186] Mice were weighted and injected IP with 0.25 ml of sterile PBS orthe indicated phosphorothioate ODN dissolved in PBS. Twenty four hourslater, spleen cells were harvested, washed, and stained for flowcytometry using phycoerythrin conjugated 6B2 to gate on B cells inconjunction with biotin conjugated anti Ly-6A/E or anti-Ia^(d)(Pharmingen, San Diego, Calif.) or anti-Bla-1 (Hardy, R. R. et al., J.Exp. Med 159:1169 (1984). Two mice were studied for each condition andanalyzed individually.

Example 6 Titration of Phosphorothioate ODN for B Cell Stimulation

[0187] B cells were cultured with phosphorothioate ODN with the sequenceof control ODN 1a or the CpG ODN 1d and 3Db and then either pulsed after20 hr with ³H uridine or after 44 hr with ³H thymidine before harvestingand determining cpm.

Example 7 Rescue of B Cells From Apoptosis

[0188] WEHI-231 cells (5×10⁴/well) were cultured for 1 hr. at 37° C. inthe presence or absence of LPS or the control ODN 1a or the CpG ODN 1dand 3Db before addition of anti-IgM (1 μ/ml). Cells were cultured for afurther 20 hr. Before a 4 hr. Pulse with 2 μCi/well ³H thymidine. Inthis experiment, cells with no ODN or anti-IgM gave 90.4×10³ cpm of ³Hthymidine incorporation by addition of anti-IgM. The phosphodiester ODNshown in Table 1 gave similar protection, though some nonspecificsuppression due to ODN degradation. Each experiment was repeated atleast 3 times with similar results.

Example 8 In vivo Induction of Murine IL-6

[0189] DBA/2 female mice (2 mos. old) were injected IP with 500 g CpG orcontrol phosphorothioate ODN. At various time points after injection,the mice were bled. Two mice were studied for each time point. IL-6 wasmeasured by ELISA, and IL-6 concentration was calculated by comparisonto a standard curve generate using recombinant IL-6. The sensitivity ofthe assay was 10 pg/ml. Levels were undetectable after 8 hrs.

Example 9 Systemic Induction of Murine IL-6 Transcription

[0190] Mice and cell lines. DBA/2, BALB/c, and C3H/HeJ mice at 5-10 wkof age were used as a source of lymphocytes. All mice were obtained fromThe Jackson Laboratory (Bar Harbor, Me.), and bred and maintained underspecific pathogen-free conditions in the University of Iowa Animal CareUnit. The mouse B cell line CH12.LX was kindly provided by Dr. G. Bishop(University of Iowa, Iowa City).

[0191] Cell preparation. Mice were killed by cervical dislocation.Single cell suspensions were prepared aseptically from the spleens frommice. T cell depleted mouse splenocytes were prepared by usinganti-Thy-1.2 and complement and centrifugation over lymphocyte M(Cedarlane Laboratories, Hornby, Ontario, Canada) as described (Krieg,A. M. et al., (1989) A role for endogenous retroviral sequences in theregulation of lymphocyte activation. J. Immunol 143:2448).

[0192] ODN and DNA. Phosphodiester oligonucleotides (O-ODN) and thebackbone modified phosphorothioate oligonucleotides (S-ODN) wereobtained from the DNA Core facility at the University of Iowa or fromOperon Technologies (Alameda, Calif.). E. Coli DNA (Strain B) and calfthymus DNA were purchased from Sigma (St. Louis, Mo.). All DNA and ODNwere purified by extraction with phenol:chloroform:isoamyl alcohol(25:24: 1) and/or ethanol precipitation. E. Coli and calf thymus DNAwere single stranded prior to use by boiling for 10 min. followed bycooling on ice for 5 min. For some experiments, E. Coli and calf thymusDNA were digested with DNase 1(2 U/μg of DNA) at 37° C. for 2 hr in1×SSC with 5 mM MgC12. To methylate the cytosine in CpG dinucleotide inE. Coli DNA, E. Coli DNA was treated with CpG methylase (M. SssI; 2 U/μgof DNA) in NEBuffer 2 supplemented with 160 μM S-adenosyl methionine andincubated overnight at 37° C. Methylated DNA was purified as above.Efficiency of methylation was confirmed by Hpa II digestion followed byanalysis by gel electrophoresis. All enzymes were purchased from NewEngland Biolabs (Beverly, Mass.). LPS level in ODN was less than 12.5ng/mg and E. Coli and calf thymus DNA contained less than 2.5 ng ofLPS/mg of DNA by Limulus assay.

[0193] Cell Culture. All cells were cultured at 37° C. in a 5% CO₂humidifier incubator maintained in RPMI-1640 supplemented with 10% (v/v)heat inactivated fetal calf serum (FCS), 1.5 mM L-glutamine, 50 μg/ml),CpG or non-CpG phosphodiester ODN (O-ODN) (20 μM), phosphorothioate ODN(S-ODN) (0.5 μM), or E. coli or calf thymus DNA (50 μg/ml) at 37° C. for24 hr. (for IL-6 production) or 5 days (for IgM production).Concentrations of stimulants were chosen based on preliminary studieswith titrations. In some cases, cells were treated with CpG O-ODN alongwith various concentrations (1-10 μg/ml) of neutralizing rat IgG1antibody against murine IL-6 (hybridoma MP5-20F3) or control rat IgG1mAB to E. Coli b-galactosidase (hybridoma GL 113; ATCC, Rockville, Md.)(20) for 5 days. At the end of incubation, culture supernatant fractionswere analyzed by ELISA as below.

[0194] In vivo induction of IL-6 and IgM. BALB/c mice were injectedintravenously (iv) with PBS, calf thymus DNA (200 μg/100 μl PBS/mouse),E. coli DNA (2001 g/100 μl PBS/mouse), or CpG or non-CpG S-ODN (200μg/100 μl PBS/mouse). Mice (two/group) were bled by retroorbitalpuncture and sacrificed by cervical dislocation at various time points.Liver, spleen, thymus, and bone marrow were removed by RNA was preparedfrom those organs using RNAzol B (Tel-Test, Friendswood, Tex.) accordingto the manufactures protocol.

[0195] ELISA. Flat-bottomed Immun 1 plates (Dynatech Laboratories, Inc.,Chantilly, Va.) were coated with 100 μl/well of anti-mouse IL-6 mAb(MP5-20F3) (2 μg/ml) or anti-mouse IgM μ-chain specific (5 μg/ml; Sigma,St. Louis, Mo.) in carbonate-bicarbonate, pH 9.6 buffer (15 nM Na₂CO₃,35 mM NaHCO₃) overnight at 4° C. The plates were then washed with TPBS(0.5 mM MgCl₂06H₂O, 2.68 mM KCl, 1.47 mM KH₂PO₄, 0.14 M NaCl, 6.6 mMK₂HPO₄, 0.5% Tween 20) and blocked with 10% FCS and TPBS for 2 hr atroom temperature and then washed again. Culture supernatants, mousesera, recombinant mouse IL-6 (Pharmigen, San Diego, Calif.) or purifiedmouse IgM (Calbiochem, San Diego, Calif.) were appropriately diluted in10% FCS and incubated in triplicate wells for 6 hr at room temperature.The plates were washed and 100 μl/well of biotinylated rat anti-mouseIL-6 monoclonal antibodies (MP5-32C 11, Pharmingen, San Diego, Calif.)(1 μg/ml in 10% FCS) or biotinylated anti-mouse Ig (Sigma, St. Louis,Mo.) were added and incubated for 45 min. at room temperature followingwashes with TPBS. Horseradish peroxidase (HRP) conjugated avidin(Bio-rad Laboratories, Hercules, Calif.) at 1:4000 dilution in 10% FCS(100 μl/well) was added and incubated at room temperature for 30 min.The plates were washed and developed with o-phenylenediaminedihydrochloride (OPD; Sigma, St. Louis Mo.) 0.05 M phosphate-citratebuffer, pH 5.0, for 30 min. The reaction was stopped with 0.67 N H₂SO₄and plates were read on a microplate reader (Cambridge Technology, Inc.,Watertown, Mass.) at 490-600 nm. The results are shown in FIGS. 1 and 2.

[0196] RT-PCR A sense primer, an antisense primer, and an internaloligonucleotide probe for IL-6 were synthesized using publishedsequences (Montgomery, R. A. and M. S. Dallman (1991), Analysis forcytokine gene expression during fetal thymic ontogeny using thepolymerase chain reaction (J. Immunol.) 147:554). cDNA synthesis andIL-6 PCR was done essentially as described by Montgomery and Dallman(Montgomery, R. A. And M. S. Dallman (1991), Analysis of cytokine geneexpression during fetal thymic ontogeny using the polymerase chainreaction (J. Immunol.) 147:554) using RT-PCR reagents from Perkin-ElmerCorp. (Hayward, Calif.). Samples were analyzed after 30 cycles ofamplification by gel electrophoresis followed by unblot analysis (stoye,J. P. et al., (1991) DNA hybridization in dried gels with fragmentedprobes: an improvement over blotting techniques, Techniques3:123).Briefly, the gel was hybridized at room temperature for 30 min. indenaturation buffer (0.05 M NaOH, 1.5M NaCl) followed by incubation for30 min. In renaturation buffer (1.5 M NaCl, 1 M Tris, pH 8) and a 30min. Wash in double distilled water. The gel was dried and prehybridizedat 47° C. for 2 hr. Hybridization buffer (5×SSPE, 0.1% SDS) containing10 μg/ml denatured salmon sperm DNA. The gel was hybridized with 2×10⁶cpm/ml g-[³² P] ATP end-labeled internal oligonucleotide probe for IL-6(5′CATTTCCACGATTTCCCA3′) SEQ ID. NO: 118) overnight at 47° C., washed 4times (2×SSC, 0.2% SDS) at room temperature and autoradiographed. Theresults are shown in FIG. 3.

[0197] Cell Proliferation assay. DBA/2 mice spleen B cells (5×10⁴cells/100 μl/well) were treated with media, CpG or non-CpG S-ODN (0.5μM) or O-ODN (20 μM) for 24 hr at 37° C. Cells were pulsed for the lastfour hr. With either [³H] Thymidine or [³H] Uridine (1 μCi/well).Amounts of [³H] incorporated were measured using Liquid ScintillationAnalyzer (Packard Instrument Co., Downers Grove, Ill.).

[0198] Transfections and CAT assays. WEHI-231 cells (10⁷ cells) wereelectroporated with 20 μg of control or human IL-6 promoter-CATconstruct (kindly provided by S. Manolagas, Univ. of Arkansas)(Pottratz, S. T. Et al., (1994) 17B-estradiol inhibits expression ofhuman interleukin-6 promotor-reporter constructs by a receptor-dependentmechanism. J. Clin. Invest. 93:944) at 250 mV and 960 μF. Cells werestimulated with various concentrations of CpG or non-CpG ODN afterelectroporation. Chloramphenicol acetyltransferase (CAT) activity wasmeasured by a solution assay (Seed, B. and J. Y. Sheen (1988) A singlephase-extraction assay for chloramphenicol acetyl transferase activity.Gene 76:271) 16 hr. after transfection. The results are presented inFIG. 5.

Example 10 Oligodeoxynucleotide Modifications Determine the Magnitude ofB Cell Stimulation by CpG Motifs

[0199] ODN were synthesized on an Applied Biosystems Inc. (Foster City,Calif.) model 380A, 380B, or 394 DNA synthesizer using standardprocedures (Beacage and Caruthers (1981) Deoxynucleosidephosphoramidites—A new class of key intermediates fordeoxypolynucleotide synthesis. Tetrahedon Letters 22, 1859-1862.).Phosphodiester ODN were synthesized using standard beta-cyanoethylphosphoramidite chemistry. Phosphorothioate linkages were introduced byoxidizing the phosphite linkage with elemental sulfur instead of thestandard iodine oxidation. The four common nucleoside phosphoramiditeswere purchased from Applied Biosystems. All phosphodiester and thioatecontaining ODN were protected by treatment with concentrated ammonia at55° C. for 12 hours. The ODN were purified by gel exclusionchromatography and lyophilized to dryness prior to use.Phosphorodithioate linkages were introduced by using deoxynucleosideS-(b-benzoylmercaptoethyl) pyrrolidino thiophosphoramidites (Wiesler, W.T. et al., (1993) In Methods in Molecular Biology: Protocols forOligonucleotides and Analogs-Synthesis and Properties, Agrawal, S.(Ed.), Humana Press, 191-206.). Dithioate containing ODN weredeprotected by treatment with concentrated ammonia at 55° C. for 12hours followed by reverse phase HPLC purification.

[0200] In order to synthesize oligomers containingmethylphosphonothioates or methylphosphonates as well as phosphodiestersat any desired internucleotide linkage, two different synthetic cycleswere used. The major synthetic differences in two cycles are thecoupling reagent where dialkylaminomethylnucleoside phosphines are usedand the oxidation reagents in the case of methylphosphonothioates. Inorder to synthesize either derivative, the condensation time has beenincreased for the dialkylaminomethylnucleoside phosphines due to theslower kinetics of coupling (Jager and Engels, (1984) Synthesis ofdeoxynucleoside methylphosphonates via a phosphonamidite approach.Tetrahedron Letters 24, 1437-1440). After the coupling step has beencompleted, the methylphosphnodiester is treated with the sulfurizingreagent (5% elemental sulfur, 100 millimolar N,N-diamethylaminopyridinein carbon disulfide/pyridine/triethylamine), four consecutive times for450 seconds each to produce methylphosphonothioates. To producemethylphosphonate linkages, the methylphosphinodiester is treated withstandard oxidizing reagent (0.1 M iodine intetrahydrofuran/2,6-lutidine/water).

[0201] The silica gel bound oligomer was treated with distilledpyridine/concentrated ammonia, 1:1, (v/v) for four days at 4 degreescentigrade. The supernatant was dried in vacuo, dissolved in water andchromatographed on a G50/50 Sephadex column.

[0202] As used herein, O-ODN refers to ODN which are phosphodiester;S-ODN are completely phosphorothioate modified; S-O=ODN are chimeric ODNin which the central linkages are phosphodiester, but the two 5′ andfive 3′ linkages are phosphorothioate modified; 2₂-O-ODN are chimericODN in which the central linkages are phosphodiester, but the two 5′ andfive 3′ linkages are phosphorodithioate modified; and MP-O-ODN arechimeric ODN in which the central linkages are phosphodiester, but thetwo 5′ and five 3′ linkages are methylphosphonate modified. The ODNsequences studied (with CpG dinucleotides indicated by underlining)include: 3D (5″ GAGAACGCTGGACCTTCCAT); (SEQ. ID. NO. 20) 3M(5′ TCCATGTCGGTCCTGATGCT); (SEQ. ID. NO. 28) 5(5′ GGCGTTATTCCTGACTCGCC); and (SEQ. ID. NO. 99) 6(5′ CCTACGTTGTATGCGCCCAGCT). (SEQ. ID NO. 100)

[0203] These sequences are representative of literally hundreds of CpGand non-CpG ODN that have been tested in the course of these studies.

[0204] Mice. DBA/2, or BXSB mice obtained from The Jackson Laboratory(Bar Harbor, Me.), and maintained under specific pathogen-freeconditions were used as a source of lymphocytes at 5-10 wk of age withessentially identical results.

[0205] Cell proliferation assay. For cell proliferation assays, mousespleen cells (5×10⁴ cells/100 μl/well) were cultured at 37° C. in a 5%CO₂ humidified incubator in RPMI-1640 supplemented with 10% (v/v) heatinactivated fetal calf serum (heated to 65° C. for experiments withO-ODN, or 56° C. for experiments using only modified ODN), 1.5 μML-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/mlstreptomycin for 24 hr or 48 hr as indicated. 1 μCi of ³H uridine orthymidine (as indicated) was added to each well, and the cells harvestedafter an additional 4 hours of culture. Filters were counted byscintillation counting. Standard deviations of the triplicate wells were<5%. The results are presented in FIGS. 6-8.

Example 11 Induction of NK Activity

[0206] Phosphodiester ODN were purchased form Operon Technologies(Alameda, Calif.). Phosphorothioate ODN were purchased from the DNA corefacility, University of Iowa, or from The Midland Certified ReagentCompany (Midland Tex.). E. coli (strain B) DNA and calf thymus DNA werepurchased from Sigma (St. Louis, Mo.). All DNA and ODN were purified byextraction with phenol:chloroform:isoamyl alcohol (25:24:1) and/orethanol precipitation. The LPS level in ODN was less than 12.5 ng/mg andE. coli and calf thymus DNA contained less than 2.5 ng of LPS/mg of DNAby Limulus assay.

[0207] Virus-free, 4-6 week old, DBA/2, C57BL/6 (B6) and congenitallythymic BALB/C mice were obtained on contract through the VeteransAffairs from the National Cancer Institute (Bethesda, Md.). C57BL/6 SCIDmice were bred in t eh SPF barrier facility at the University of IowaAnimal Care Unit.

[0208] Human peripheral monucluclear blood leukocytes (PBMC) wereobtained as previously described (Ballas, Z. K. et al., (1990) J.Allergy Clin. Immunol. 85:453; Ballas, Z. K. And W. Rasmussen (1990) J.Immunol. 145:1039; Ballas, Z. K. and W. Rasmussen (1993) J. Immunol.150;17). Human or murine cells were cultured at 5×10⁶/well, at 37° C. ina 5% CO₂ humidified atmosphere in 24-well plates (Ballas, Z. K. Et al.,(1990) J. Allergy Clin. Immunol. 85:453; Ballas, Z. K. And W. Rasmussen(1990) J. Immunol 145:1039; and Ballas, Z. K. and W. Rasmussen (1193) J.Immunol, 150:17), with medium alone or with CpG or non-CpG ODN at theindicated concentrations, or with E. coli or calf thymus (50 μg/ml) at37° C. for 24 hr. All cultures were harvested at 18 hr. and the cellswere used as effectors in a standard 4 hr. ⁵¹Cr-release assay againstK562 (human) or YAC-1 (mouse) target cells as previously described. Forcalculation of lytic units (LU), 1 LU was defined as the number of cellsneeded to effect 30% specific lysis. Where indicated, neutralizingantibodies against IFN-β (Lee Biomolecular, San Diego, Calif.) or IL-12(C15.1, C15.6, C17.8, and C17.15; provided by Dr. Giorgio Trinchieri,The Winstar Institute, Philadelphia, Pa.) or their isotype controls wereadded at the initiation of cultures to a concentration of 10 μg/ml. Foranti-IL-12 additional, 10 μg of each of the 4 MAB (or isotype controls)were added simultaneously. Recombinant human IL-2 was used at aconcentration of 100 U/ml.

Example 12 Prevention of the Development of an Inflammatory CellularInfiltrate and Eosinophilia in a Murine Model of Asthma

[0209] 6-8 week old C56BL/6 mice (from The Jackson Laboratory, BarHarbor, Me.) were immunized with 5,000 Schistosoma mansoni eggs byintraperitoneal (i.p.) injection on days 0 and 7. Schistosoma mansonieggs contain an antigen (Schistosoma mansoni egg antigen (SEA)) thatinduces a Th2 immune response (e.g. production of IgE antibody). IgEantibody production is known to be an important cause of asthma.

[0210] The immunized mice were then treated with oligonucleotides (30 μgin 200 μl saline by i.p. injection), which either contained anunmethylated CpG motif (i.e., TCCATGACGTTCCTGACGTT; SEQ ID NO.10) or didnot (i.e., control, TCCATGAGCTTCCTGAGTCT; SEQ ID NO. 8). Soluble SeEA(10 μg in 25 μl of saline) was administered by intranasal instillationon days 14 and 21. Saline was used as a control.

[0211] Mice were sacrificed at various times after airway challenge.Whole lung lavage was performed to harvest airway and alveolarinflammatory cells. Cytokine levels were measured from lavage fluid byELISA. RNA was isolated from whole lung for Northern analysis and RT-PCRstudies using CsC1 gradients. Lungs were inflated and perfused with 4%paraformaldehyde for histologic examination.

[0212]FIG. 9 shows that when the mice are initially injected with theeggs i.p., and then inhale the egg antigen (open circle), manyinflammatory cells are present in the lungs. However, when the mice areinitially given a nucleic acid containing an unmethylated CpG motifalong with the eggs, the inflammatory cells in the lung are notincreased by subsequent inhalation of the egg antigen (open triangles).

[0213]FIG. 10 shows that the same results are obtained only wheneosinophils present in the lung lavage are measured. Eosinophils are thetype of inflammatory cell most closely associated with asthma.

[0214]FIG. 11 shows that when the mice are treated with a control oligoat the time of the initial exposure to the egg, there is little effecton the subsequent influx of eosinophils into the lungs after inhalationof SEA. Thus, when mice inhale the eggs on days 14 or 21, they developan acute inflammatory response in the lungs. However, giving a CpG oligoalong with the eggs at the time of initial antigen exposure on days 0and 7 almost completely abolishes the increase in eosinophils when themice inhale the egg antigen on day 14.

[0215]FIG. 12 shows that very low doses of oligonucleotide (<10 μg) cangive this protection.

[0216]FIG. 13 shows that the resultant inflammatory response correlateswith the levels of the Th2 cytokine IL-4 in the lung.

[0217]FIG. 14 shows that administration of an oligonucleotide containingan unmethylated CpG motif can actually redirect the cytokine response ofthe lung to production of IL-12, indicating the Th1 type of immuneresponse.

[0218]FIG. 15 shows that administration of an oligonucleotide containingan unmethylated CpG motif can also redirect the cytokine response of thelung to production of IFN-γ, indicating a Th1 type of immune response.

Example 13 CpG Oligonucleotides Induce Human PBMC to Secrete Cytokines.

[0219] Human PBMC were prepared from whole blood by standardcentrifugation over Ficoll hypaque. Cells (5×10⁵/ml) were cultured in10% autologous serum in 95 well microtiter plates with CpG or controloligodeoxynucleotides (24 μg/ml for phosphodiester oligonucleotides; 6g/ml for nuclease resistant phosphorothioate oligonucleotides) for 4 hrin the case of TNF-α or 24 hr. For the other cytokines beforesupernatant harvest and assay, measured by ELISA using Quantikine kitsor reagents from R&D Systems (pg/ml) or cytokine ELISA kits fromBiosource (for IL-12 assay). Assays were performed as per themanufacturer's instructions. Data are presented in Table 6 as the levelof cytokine above that in wells with no added oligodeoxynucleotide.

[0220] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents of thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 123 1 20 DNA Artificial Sequence Synthetic Oligonucleotide 1atggaaggtc cagcgttctc 20 2 20 DNA Artificial Sequence SyntheticOligonucleotide 2 atcgacctac gtgcgttctc 20 3 20 DNA Artificial SequenceSynthetic Oligonucleotide 3 tccataacgt tcctgatgct 20 4 15 DNA ArtificialSequence Synthetic Oligonucleotide 4 gctagatgtt agcgt 15 5 19 DNAArtificial Sequence Synthetic Oligonucleotide 5 gagaacgtcg accttcgat 196 15 DNA Artificial Sequence Synthetic Oligonucleotide 6 gcatgacgttgagct 15 7 20 DNA Artificial Sequence Synthetic Oligonucleotide 7tccatgacgt tcctgatgct 20 8 20 DNA Artificial Sequence SyntheticOligonucleotide 8 tccatgagct tcctgagtct 20 9 20 DNA Artificial SequenceSynthetic Oligonucleotide 9 tccaagacgt tcctgatgct 20 10 20 DNAArtificial Sequence Synthetic Oligonucleotide 10 tccatgacgt tcctgacgtt20 11 21 DNA Artificial Sequence Synthetic Oligonucleotide 11 tccatgagcttcctgagtgc t 21 12 20 DNA Artificial Sequence Synthetic Oligonucleotide12 ggggtcaacg ttgagggggg 20 13 15 DNA Artificial Sequence SyntheticOligonucleotide 13 gctagangtt agcgt 15 14 15 DNA Artificial SequenceSynthetic Oligonucleotide 14 gctagacgtt agngt 15 15 20 DNA ArtificialSequence Synthetic Oligonucleotide 15 atcgactctc gagcgttctc 20 16 20 DNAArtificial Sequence Synthetic Oligonucleotide 16 atngactctn gagngttctc20 17 20 DNA Artificial Sequence Synthetic Oligonucleotide 17 atngactctcgagcgttctc 20 18 20 DNA Artificial Sequence Synthetic Oligonucleotide 18atcgactctc gagcgttntc 20 19 20 DNA Artificial Sequence SyntheticOligonucleotide 19 atggaaggtc caacgttctc 20 20 20 DNA ArtificialSequence Synthetic Oligonucleotide 20 gagaacgctg gaccttccat 20 21 20 DNAArtificial Sequence Synthetic Oligonucleotide 21 gagaacgctc gaccttccat20 22 20 DNA Artificial Sequence Synthetic Oligonucleotide 22 gagaacgctcgaccttcgat 20 23 20 DNA Artificial Sequence Synthetic Oligonucleotide 23gagcaagctg gaccttccat 20 24 20 DNA Artificial Sequence SyntheticOligonucleotide 24 gagaangctg gaccttccat 20 25 20 DNA ArtificialSequence Synthetic Oligonucleotide 25 gagaacgctg gacnttccat 20 26 20 DNAArtificial Sequence Synthetic Oligonucleotide 26 gagaacgatg gaccttccat20 27 20 DNA Artificial Sequence Synthetic Oligonucleotide 27 gagaacgctccagcactgat 20 28 20 DNA Artificial Sequence Synthetic Oligonucleotide 28tccatgtcgg tcctgatgct 20 29 20 DNA Artificial Sequence SyntheticOligonucleotide 29 tccatgctgg tcctgatgct 20 30 20 DNA ArtificialSequence Synthetic Oligonucleotide 30 tccatgtngg tcctgatgct 20 31 20 DNAArtificial Sequence Synthetic Oligonucleotide 31 tccatgtcgg tnctgatgct20 32 20 DNA Artificial Sequence Synthetic Oligonucleotide 32 tccatgtcggtcctgctgat 20 33 20 DNA Artificial Sequence Synthetic Oligonucleotide 33tccatgccgg tcctgatgct 20 34 20 DNA Artificial Sequence SyntheticOligonucleotide 34 tccatggcgg tcctgatgct 20 35 20 DNA ArtificialSequence Synthetic Oligonucleotide 35 tccatgacgg tcctgatgct 20 36 20 DNAArtificial Sequence Synthetic Oligonucleotide 36 tccatgtcga tcctgatgct20 37 20 DNA Artificial Sequence Synthetic Oligonucleotide 37 tccatgtcgctcctgatgct 20 38 20 DNA Artificial Sequence Synthetic Oligonucleotide 38tccatgtcgt tcctgatgct 20 39 20 DNA Artificial Sequence SyntheticOligonucleotide 39 tccatgacgt ccctgatgct 20 40 20 DNA ArtificialSequence Synthetic Oligonucleotide 40 tccatcacgt gcctgatgct 20 41 19 DNAArtificial Sequence Synthetic Oligonucleotide 41 ggggtcagtc ttgacgggg 1942 15 DNA Artificial Sequence Synthetic Oligonucleotide 42 gctagacgttagtgt 15 43 15 DNA Artificial Sequence Synthetic Oligonucleotide 43gctagacntt agtgt 15 44 20 DNA Artificial Sequence SyntheticOligonucleotide 44 tccatgtngt tcctgatgct 20 45 18 DNA ArtificialSequence Synthetic Oligonucleotide 45 tctcccagcg tgcgccat 18 46 24 DNAArtificial Sequence Synthetic Oligonucleotide 46 tcgtcgtttt gtcgttttgtcgtt 24 47 20 DNA Artificial Sequence Synthetic Oligonucleotide 47tcgtcgttgt cgttgtcgtt 20 48 21 DNA Artificial Sequence SyntheticOligonucleotide 48 tgtcgtttgt cgtttgtcgt t 21 49 22 DNA ArtificialSequence Synthetic Oligonucleotide 49 tcgtcgttgt cgttttgtcg tt 22 50 19DNA Artificial Sequence Synthetic Oligonucleotide 50 tgtcgttgtcgttgtcgtt 19 51 14 DNA Artificial Sequence Synthetic Oligonucleotide 51tcgtcgtcgt cgtt 14 52 20 DNA Artificial Sequence SyntheticOligonucleotide 52 tcctgtcgtt ccttgtcgtt 20 53 20 DNA ArtificialSequence Synthetic Oligonucleotide 53 tcctgtcgtt ttttgtcgtt 20 54 21 DNAArtificial Sequence Synthetic Oligonucleotide 54 tcgtcgctgt ctgcccttct t21 55 21 DNA Artificial Sequence Synthetic Oligonucleotide 55 tcgtcgctgttgtcgtttct t 21 56 21 DNA Artificial Sequence Synthetic Oligonucleotide56 gcgtgcgttg tcgttgtcgt t 21 57 6 DNA Artificial Sequence SyntheticOligonucleotide 57 gtcgtt 6 58 6 DNA Artificial Sequence SyntheticOligonucleotide 58 gtcgct 6 59 24 DNA Artificial Sequence SyntheticOligonucleotide 59 accatggacg atctgtttcc cctc 24 60 18 DNA ArtificialSequence Synthetic Oligonucleotide 60 taccgcgtgc gaccctct 18 61 24 DNAArtificial Sequence Synthetic Oligonucleotide 61 accatggacg aactgtttcccctc 24 62 24 DNA Artificial Sequence Synthetic Oligonucleotide 62accatggacg agctgtttcc cctc 24 63 24 DNA Artificial Sequence SyntheticOligonucleotide 63 accatggacg acctgtttcc cctc 24 64 24 DNA ArtificialSequence Synthetic Oligonucleotide 64 accatggacg tactgtttcc cctc 24 6524 DNA Artificial Sequence Synthetic Oligonucleotide 65 accatggacggtctgtttcc cctc 24 66 24 DNA Artificial Sequence SyntheticOligonucleotide 66 accatggacg ttctgtttcc cctc 24 67 15 DNA ArtificialSequence Synthetic Oligonucleotide 67 cacgttgagg ggcat 15 68 15 DNAArtificial Sequence Synthetic Oligonucleotide 68 ctgctgagac tggag 15 6912 DNA Artificial Sequence Synthetic Oligonucleotide 69 tcagcgtgcg cc 1270 17 DNA Artificial Sequence Synthetic Oligonucleotide 70 atgacgttcctgacgtt 17 71 17 DNA Artificial Sequence Synthetic Oligonucleotide 71tctcccagcg ggcgcat 17 72 18 DNA Artificial Sequence SyntheticOligonucleotide 72 tctcccagcg cgcgccat 18 73 20 DNA Artificial SequenceSynthetic Oligonucleotide 73 tccatgtcgt tcctgtcgtt 20 74 20 DNAArtificial Sequence Synthetic Oligonucleotide 74 tccatagcgt tcctagcgtt20 75 21 DNA Artificial Sequence Synthetic Oligonucleotide 75 tcgtcgctgtctccgcttct t 21 76 19 DNA Artificial Sequence Synthetic Oligonucleotide76 tcctgacgtt cctgacgtt 19 77 19 DNA Artificial Sequence SyntheticOligonucleotide 77 tcctgtcgtt cctgtcgtt 19 78 20 DNA Artificial SequenceSynthetic Oligonucleotide 78 tccatgtcgt ttttgtcgtt 20 79 20 DNAArtificial Sequence Synthetic Oligonucleotide 79 tccaggactt ctctcaggtt20 80 20 DNA Artificial Sequence Synthetic Oligonucleotide 80 tccatgcgtgcgtgcgtttt 20 81 20 DNA Artificial Sequence Synthetic Oligonucleotide 81tccatgcgtt gcgttgcgtt 20 82 20 DNA Artificial Sequence SyntheticOligonucleotide 82 tccacgacgt tttcgacgtt 20 83 20 DNA ArtificialSequence Synthetic Oligonucleotide 83 gcggcgggcg gcgcgcgccc 20 84 25 DNAArtificial Sequence Synthetic Oligonucleotide 84 tgtcgttgtc gttgtcgttgtcgtt 25 85 13 DNA Artificial Sequence Synthetic Oligonucleotide 85tgtcgttgtc gtt 13 86 20 DNA Artificial Sequence SyntheticOligonucleotide 86 tccacgacgt tttcgacgtt 20 87 20 DNA ArtificialSequence Synthetic Oligonucleotide 87 tccatgacga tcctgatgct 20 88 20 DNAArtificial Sequence Synthetic Oligonucleotide 88 tccatgacgc tcctgatgct20 89 15 DNA Artificial Sequence Synthetic Oligonucleotide 89 gctagacgttagcgt 15 90 8 DNA Artificial Sequence Synthetic Oligonucleotide 90tcaacgtt 8 91 8 DNA Artificial Sequence Synthetic Oligonucleotide 91tcaagctt 8 92 8 DNA Artificial Sequence Synthetic Oligonucleotide 92tcagcgct 8 93 8 DNA Artificial Sequence Synthetic Oligonucleotide 93tcatcgat 8 94 8 DNA Artificial Sequence Synthetic Oligonucleotide 94tcttcgaa 8 95 8 DNA Artificial Sequence Synthetic Oligonucleotide 95ccaacgtt 8 96 8 DNA Artificial Sequence Synthetic Oligonucleotide 96tcaacgtc 8 97 20 DNA Artificial Sequence Synthetic Oligonucleotide 97tccaggactt tcctcaggtt 20 98 20 DNA Artificial Sequence SyntheticOligonucleotide 98 ttcaggactt tcctcaggtt 20 99 20 DNA ArtificialSequence Synthetic Oligonucleotide 99 ggcgttattc ctgactcgcc 20 100 22DNA Artificial Sequence Synthetic Oligonucleotide 100 cctacgttgtatgcgcccag ct 22 101 7 DNA Artificial Sequence Synthetic Oligonucleotide101 tgtcgct 7 102 7 DNA Artificial Sequence Synthetic Oligonucleotide102 tgtcgtt 7 103 7 DNA Artificial Sequence Synthetic Oligonucleotide103 tgacgtc 7 104 8 DNA Artificial Sequence Synthetic Oligonucleotide104 tgacgtca 8 105 6 DNA Artificial Sequence Synthetic Oligonucleotide105 aacgtt 6 106 7 DNA Artificial Sequence Synthetic Oligonucleotide 106caacgtt 7 107 8 DNA Artificial Sequence Synthetic Oligonucleotide 107aacgttct 8 108 7 DNA Artificial Sequence Synthetic Oligonucleotide 108tgacgtt 7 109 6 DNA Artificial Sequence Synthetic Oligonucleotide 109gccggt 6 110 6 DNA Artificial Sequence Synthetic Oligonucleotide 110gacggt 6 111 6 DNA Artificial Sequence Synthetic Oligonucleotide 111gacgtc 6 112 6 DNA Artificial Sequence Synthetic Oligonucleotide 112cacgtg 6 113 7 DNA Artificial Sequence Synthetic Oligonucleotide 113cgacgtt 7 114 20 DNA Artificial Sequence Synthetic Oligonucleotide 114atggaaggtc cagtgttctc 20 115 20 DNA Artificial Sequence SyntheticOligonucleotide 115 atggactctc cagcgttctc 20 116 20 DNA ArtificialSequence Synthetic Oligonucleotide 116 atcgactctc gagngttctc 20 117 15DNA Artificial Sequence Synthetic Oligonucleotide 117 gctagangtt agtgt15 118 18 DNA Artificial Sequence Synthetic Oligonucleotide 118catttccacg atttccca 18 119 21 DNA Artificial Sequence SyntheticOligonucleotide 119 tcgtcgctgt ctgcccttct t 21 120 21 DNA ArtificialSequence Synthetic Oligonucleotide 120 tcgtcgctgt tgtcgtttct t 21 121 20DNA Artificial Sequence Synthetic Oligonucleotide 121 tccttgtcgttcctgtcgtt 20 122 20 DNA Artificial Sequence Synthetic Oligonucleotide122 tccatgtngt tcctgtngtt 20 123 23 DNA Artificial Sequence SyntheticOligonucleotide 123 tcgtcgtttt gtcgttttgt cgt 23

1. An isolated nucleic acid sequence containing at least oneunmethylated CpG dinucleotide and having a formula: 5′N ₁ X ₁ CGX ₂ N₂3′ wherein at least one nucleotide separates consecutive CpGs; X₁ isadenine, guanine, or thymine; X₂ is cytosine or thymine; N is anynucleotide and N₁+N₂ is from about 0-26 bases with the proviso thatN₁+N₂ does not contain a CCGG quadmer or more than one CCG or CGGtrimer; and the nucleic acid sequence is from about 8-30 bases inlength.
 2. The nucleic acid sequence of claim 1, wherein X₁ is thymine3. The nucleic acid sequence of claim 1, wherein X₂ is thymine.
 4. Thenucleic acid sequence of claim 1, which is GTCG (T/C) T or TGACGTT. 5.The nucleic acid sequence of claim 1, wherein the sequence is TGTCG(T/C) T.
 6. The nucleic acid sequence of claim 1, which isTCCATGTCGTTCCTGTCGTT.
 7. The nucleic acid sequence of claim 1, which isTCCTGACGTTCCTGACGTT.
 8. The nucleic acid sequence of claim 1, which isTCGTCGTTTTGTCGTTTTGTCGTT.
 9. An isolated nucleic acid sequencecontaining at least one unmethylated CpG dinucleotide and having theformula: 5′NX ₁ X ₂ CGX ₃ X ₄ N3′wherein at least one nucleotideseparates consecutive CpGs; X₁X₂ is selected from the group consistingof GpT, GpG, GpA, ApT and ApA; X₃X₄ is selected from the groupconsisting of TpT or CpT; N is any nucleotide and N₁N₂ is from about0-26 bases with the proviso that N₁ and N₂ does not contain a CCGGquadmer or more than one CCG or CGG trimer; and the nucleic acidsequence is from about 8-30 bases in length.
 10. The nucleic acidsequence of claim 9, wherein the nucleotide that separates at least twoconsecutive CpGs is thymine.
 11. The nucleic acid sequence of claim 9,wherein X₃ and X₄ are thymine.
 12. A nucleic acid sequence of any ofclaims 1 or 9, wherein at least one nucleotide has a phosphate backbonemodification.
 13. The nucleic acid sequence of claim 12, wherein thephosphate backbone modification is a phosphorothioate orphosphorodithioate modification.
 14. The nucleic acid sequence of claim13, wherein the phosphate backbone modification occurs at the 5′ end ofthe nucleic acid.
 15. The nucleic acid sequence of claim 14, wherein themodification occurs at the first two internucleotide linkages of the 5′end of the nucleic acid.
 16. The nucleic acid sequence of claim 13,wherein the phosphate backbone modification occurs at the 3′ end of thenucleic acid.
 17. The nucleic acid sequence of claim 16, wherein themodification occurs at the last five internucleotide linkages of the 3′end of the nucleic acid.
 18. A method of stimulating immune activationin a subject, wherein the stimulation is predominantly a Th1 pattern ofimmune activation, comprising administering to the subject a nucleicacid sequence having the formula of claim 1 or claim
 9. 19. The methodof claim 18, wherein the subject is human.
 20. A method of stimulatingcytokine production in a subject comprising administering to the subjecta nucleic acid sequence having the formula of claim 1 or claim
 9. 21.The method of claim 20, wherein the cytokine is selected from the groupconsisting of: IL-6, IL-12, IFN-γ, TNF-α and GM-CSF.
 22. The method ofclaim 20, wherein the subject is human.
 23. The method of claim 20,where the nucleic acid sequence is selected from the group consistingof: TCCATGTCGCTCCTGATGCT, TCCATAACGTTCCTGATGCT, TCCATGACGATCCTGATGCT,TCCATGGCGGTCCTGATGCT, TCCATGTCGGTCCTGATGCT, TCCATAACGTCCCTGATGCT,TCCATGTCGTTCCTGATGCT; and TCGTCGTTTTGTCGTTTTGTCGTT.


24. A method of stimulating NK lytic activity in a subject comprisingadministering to the subject a nucleic acid sequence having the formulaof claim 1 or claim
 9. 25. The method of claim 24, where the subject ishuman.
 26. The method of claim 24, where the nucleic acid sequence isselected from the group consisting of: TCGTCGTTGTCGTTGTCGTT,TCCATGACGGTCCTGATGCT, TCCATGACGATCCTGATGCT, TCCATGACGCTCCTGATGCT,TCCATGACGTTCCTGATGCT, TCCATAACGTTCCTGATGCT, TCCATCACGTGCCTGATGCT,GGGGTCAACGTTGAGGGGGG, TCGTCGTTTTGTCGTTTTGTCGTT, TCGTCGTTGTCGTTTTGTCGTT,GCGTGCGTTGTCGTTGTCGTT, TGTCGTTTGTCGTTTGTCGTT, TGTCGTTGTCGTTGTCGTT; andTCGTCGTCGTCGTT.


27. A method of stimulating B cell proliferation in a subject,comprising administering to the subject a nucleic acid sequence havingthe formula of claim 1 or claim
 9. 28. The method of claim 27, where thesubject is human.
 29. The method of claim 27, where the nucleic acidsequence is selected from the group consisting of:TCCTGTCGTTCCTTGTCGTT), TCCTGTCGTTTTTTGTCGTT, TCGTCGCTGTCTGCCCTTCTT,TCGTCGCTGTTGTCGTTTCTT, TCGTCGTTTTGTCGTTTTGTCGTT, TCGTCGTTGTCGTTTTGTCGTT;and TGTCGTTGTCGTTGTCGTT.


30. A method of stimulating immune activation in a subject comprisingadministering to a subject an nucleic acid sequence having the formulaof claim 1, wherein the nucleic acid sequence acts as an adjuvant. 31.The method of claim 30, where the subject is a mammal.
 32. The method ofclaim 30, where the nucleic acid sequence is selected from the groupconsisting of: TCCATGACGTTCCTGACGTT, GTCG (T/C) T; and TGTCG (T/C) T.


33. A method for treating a subject having an asthmatic disorder byadministering to the subject an nucleic acid sequence in apharmaceutically acceptable carrier having the formula of claim
 1. 34.The method of claim 33, where the subject is human.
 35. The method ofclaim 33, where the nucleic acid sequence is TCCATGACGTTCCTGACGTT.
 36. Amethod for treating a subject having an autoimmune or other CpGassociated disorder by inhibiting CpG-mediated leukocyte activationcomprising administering to the subject an inhibitor of endosomalacidification in a pharmaceutically acceptable carrier.
 37. The methodof claim 36, where the subject is human.
 38. The method of claim 36,where the inhibitor is selected from the group consisting of:bafilomycin A, chloroquine, and monensin.
 39. The method of claim 38,where the inhibitor is administered at a dosage of the less than about10 μM.
 40. The method of claim 36, wherein the disorder is selected fromthe group consisting of systemic lupus erythematosus, sepsis,inflammatory bowel disease, psoriasis, gingivitis, arthritis, Crohn'sdisease, Grave's disease and asthma.
 41. The method of claim 40, wherethe disorder is systemic lupus erythematosus.